BACKGROUND
the apiaceae family  contains a number of important vegetable and spice crop species including carrot, celery, fennel, cilantro, parsley and parsnip.
carrot  is the most economically important species in the apiaceae, with more than  <dig> million tons produced yearly worldwide .
carrot consumption has steadily increased in recent decades for several reasons, including a heightened awareness of its health-promoting attributes , development of fresh-cut carrot products convenient for consumers, and adaptation of improved cultivars for warmer production areas  <cit> .
despite their economic importance, carrot and other apiaceae have relatively underdeveloped molecular resources  <cit> .
three unsaturated linkage maps, mainly based on anonymous dominant aflp markers, have been constructed in carrot  <cit>  to assist breeding of this species.
inheritance studies on natural carotenoid mutants have identified factors conditioning root pigment accumulation  <cit> , and both simply inherited pigment traits and qtl have been included in these maps  <cit> .
the more recent addition of  <dig> genes from the carotenoid biosynthetic pathway  <cit> , as well as transposon-display markers  <cit> , onto one of the maps has increased both maker informativeness and coverage.
the latter map, which was constructed using an f <dig> family derived from a cultivated × wild carrot cross, is considered the carrot reference map because it harbors important phenotypic traits, has fairly good coverage and includes the largest number of informative markers.
however, direct comparisons between the reference and other carrot maps are currently difficult due to the lack of common markers across maps.
this fact seriously limits the usefulness of these maps for assisting breeding, especially considering that a number of important carrot traits, including nematode resistance  <cit> , anthocyanin pigmentation and reduced-sugar accumulation  <cit> , have been mapped in unrelated genetic backgrounds.
the lack of common markers across carrot maps is mainly due to an insufficient availability of informative and robust pcr-based markers.
the carotenoid genes mapped by just et al.
<cit>  are not easily transferred to other maps with different genetic backgrounds, since they were mainly mapped by sequence detection of single polymorphism nucleotides , rather than by fragment length polymorphisms, and because sequence conservation was very high  in some carotenoid genes  <cit> .
thus, for these genes, little snp polymorphism may be expected.
the development of polymorphic and robust pcr-based markers in carrot, such as microsatellites, would facilitate their inclusion in different maps, thus serving as anchoring points for map integration.
this would immediately increase map marker density, ssr-tagging relevant phenotypic traits and, perhaps, facilitate applications such as positional gene-cloning.
in addition, other carrot genetic research studies, such as analysis of genetic diversity and phylogenetic reconstructions, previously approached using anonymous dominant aflp markers  <cit>  or laborious time-consuming codominant rflps  <cit> , would also benefit from the development of microsatellite markers.
microsatellites, or simple sequence repeats , are the marker of choice in many molecular genetic applications including mapping, fingerprinting, genetic diversity, population structure analysis, gene flow and germplasm conservation studies.
their widespread adoption is due to several desirable characteristics: they are codominant, frequently and evenly distributed throughout genomes, selectively neutral, highly reproducible and rely on simple polymerase chain reaction  technology.
in addition they are ubiquitous  and hypervariable.
the latter property is attributed to a high mutation rate of these repeats resulting from dna polymerase slippage during dna synthesis  <cit> .
this mutational mechanism generates gains or losses of one or a few repeat units in the microsatellite, which accumulate more rapidly than point mutations and indels  <cit> , leading to a high number of alleles per locus.
in plants, the high polymorphism found in microsatellites has allowed the detection of variability in species otherwise characterized by low levels of genetic diversity  <cit> .
in apiaceae, very few publicly available ssrs have been reported previously, and these were developed from carrot  and celery , the two most economically important species in the family.
the availability of a large set of ssrs in carrot is likely to benefit research in other apiaceae, since significant marker transferability has been observed across related taxa  <cit> .
this is of particular interest to research groups working in minor crops or species with limited research funds; many laboratories have sufficient resources and expertise for running ssr-based pcr analyses, although perhaps not for the isolation and characterization of new loci.
in this study we report on the development of  <dig> new carrot ssr markers.
further characterization of these loci includes analysis of ssr distributions in genomic and est sequence, linkage mapping onto the carrot reference map, evaluation of their mapping potential in subsets of seven carrot f <dig> mapping populations, evaluation of their potential for assessing genetic diversity among daucus carota accessions, and evaluation of ssr marker transferability across  <dig> apiaceae taxa.
RESULTS
distribution of microsatellites in genomic and est sequence
substantial variation in ssr frequency and distribution between genomic and est sequence of carrot was found .
because frequency distributions for gssrs mainly reflect the library enrichment procedure , conditioning the type, length and sequence motifs of the ssrs obtained, only ssrs from bac end sequence  were used as representatives of genomic ssrs in comparisons with ssrs from est sequence .
overall, ssrs occurred at a lower density in genomic dna   than in ests .
dinucleotide , trinucleotide , and tetranucleotide repeats  predominated in genomic dna, whereas in ests trinucleotides were the predominant repeats accounting for 50% of the total ssrs.
the absolute density for this repeat type in est data  was nearly twice its density found in genomic sequence .
the same was observed for dinucleotide and hexanucleotide repeats, which were more than twice as frequent in ests  compared to genomic dna  .
conversely, the density of pentanucleotides, heptanucleotides and octanucleotides was more than  <dig> fold higher in genomic sequence than in est sequence.
* a minimum of  <dig> repeat units  for dinucleotides,  <dig> r.u.
for trinucleotides, and  <dig> r.u.
for tetra-, penta-, hexa- hepta- and octanucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot.
ǂ only density values for ssrs from bac end sequences  and from ests  are presented .
‡ ssr motif considering complementary ; ssr motifs occurring at a rate of at least 5% of the total are listed with occurrence as percentage within each ssr type class in parentheses; ssr classes without predominance of a particular motif are denoted as "-".
ζ total length of sequences analyzed.
γ includes both gssrs and bssrs.
comparisons among microsatellites isolated from the different sequence datasets  revealed that di-, tri-, and tetranucleotide repeats accounted for  <dig> % to  <dig> % of the ssrs among the three types evaluated, with di- and tetranucleotide motifs accounting for most of the gssrs, while trinucleotide motifs were most common among bssrs and essrs .
within genomic dna, variation was also found between the two sequence datasets examined.
overall, gssrs had more repeat units  and consequently were longer  than bssrs .
the larger number of repeat units in gssrs compared to bssrs was evident and significant  for di, tri, and tetranucleotides, whereas penta to octanucleotides had the same mean number of repeats in both datasets.
ssr length had a similar relationship for these repeat types in both datasets.
with regard to repeat types, gssrs yielded a higher proportion of di and tetranucleotides, as compared to bssrs, whereas trinucleotides and penta to octanucleotides were more frequent in the latter group.
in addition, gssrs included a significant fraction  of often long, compound microsatellites, such repeats being nearly  <dig> times more frequent in this group compared to bssrs.
based upon posterior probabilities, the distributions of sequence motifs n, n, n, etc.
; n, n, n, etc.)
in the dinucleotide and trinucleotide classes were not random for bssrs and essrs .
in bac end sequence, n and n dinucleotides were more and less frequent than expected, respectively.
in ests, n repeats were less frequent than expected whereas n and n dinucleotides occurred at a higher-than-expected incidence.
ag and ac motifs occurred frequently in dinucleotide ssrs of all sequence origins, although at dinucleotides were most frequent among bssrs.
repeats of aag, act, and aaat were abundant and common to both genomic and essrs.
on the other hand, repeats of aac, agt, acat, aatt, and aaaag, predominated mainly among genomic microsatellites, whereas agg, agc, aaag, agccc, aaaaag, and aaagag motifs were most frequent in est ssrs .
comparisons between observed and expected trinucleotide repeat motifs presented no clear trends but observed distributions differed from those expected for many motifs to result in significant deviation based upon chi-square analysis .
*a minimum of  <dig> repeat units  for dinucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot.
chi-squared distributions indicate comparison of observed ssr distribution of ssr motif types n, n, n, etc.
; n, n, n, etc.)
to posterior probabilities.
tests are partitioned by library and sequence motif.
χ <dig> = chi-square statistic; df = degrees of freedom.
ssrs were non-randomly distributed across motif types.
microsatellite distribution was not uniform across coding and non-coding regions of carrot.
frequency distributions of both repeat types and sequence motifs for each microsatellite origin, i.e., a library enrichment procedure , bac end-derived  and est-derived ssrs , varied markedly across these dna fractions .
among gssrs, di- and tetranucleotide repeats were most common, and tetranucleotide repeats were distinctive in being the only gssr repeat type with a significantly different occurrence inside and outside of orfs, with over 70% of tetranucleotide ssrs inside orfs.
analysis of repeats in bac end sequence  revealed a predominance of trinucleotides in coding sequences,  compared to non coding regions, whereas tetranucleotides were somewhat more abundant in non-coding regions.
the overrepresentation of trinucleotides in orfs of genomic dna was higher than expected by chi-square analysis  and associated to a high frequency of aag, aac, agt, act, acg, and acc motifs, whereas non-coding regions rich in tetranucleotides were particularly gc-poor with an abundance of aaat and aatt motifs .
dinucleotide, pentanucleotide, and hexanucleotide repeats were nearly equally frequent in protein coding and non-coding regions of bac end sequence .
* a minimum of  <dig> repeat units  for dinucleotides,  <dig> r.u.
for trinucleotides, and  <dig> r.u.
for tetra-, penta-, hexa-, hepta-,and octanucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot.
chi-square distributions indicate observed ssr motif types inside versus outside orfs as compared to posterior probabilities.
χ <dig> = chi-squared statistic; df = degrees of freedom.
observed distributions generally fit those expected  except for tetranucleotides for gssrs , and trinucleotides for bssrs .
for essrs  the observed distributions only fit expected ones for tri-, penta-, and hexanucleotide repeats, with observed occurrence of di- and tetranucleotide repeats occurring much more often outside orfs than expected.
the distribution of ssr types in est sequences was especially variable, with trinucleotides predominating in orfs, representing more than 65% of the ssrs found inside orfs  and 26% of all essrs, and dinucleotides and tetranucleotides predominating in non-protein coding sequences of the ests  .
in est orfs, the most frequent trinucleotide motifs were aag, act, agg, agc, and acc.
in the dinucleotides-rich utr region of ests, ag and ac motifs were three times more frequent than in protein-coding regions of ests .
ssr marker development
primer pairs for  <dig> gssr and  <dig> bssr loci were designed.
of these  <dig> primers pairs,  <dig> flanked single ssrs  and  <dig> flanked multiple ssrs .
markers with single perfect repeats included  <dig> dinucleotides,  <dig> trinucleotides,  <dig> tetranucleotides,  <dig> pentanucleotides,  <dig> hexanucleotides,  <dig> heptanucleotides, and  <dig> each of mono, octa and nonanucleotides.
further information on these  <dig> ssr markers is presented in additional file  <dig> - table s <dig>  including primer sequence, annealing temperature, repeat motif and its position in template sequence, expected amplicon length, and the template dna sequence carrying the ssr .
for a number of microsatellites detected computationally, we were not able to design primers because they either lacked suitable flanking sequences or the total sequence length was too short .
marker polymorphism analyses in carrot f <dig> families
a total of  <dig> ssrs  were successfully characterized in subsets of  <dig> carrot f <dig> families .
details on the performance of each ssr marker in the different mapping populations are presented in additional file  <dig> - table s <dig>  overall,  <dig> ssrs  were polymorphic in at least one mapping population.
these included  <dig> gssrs and  <dig> bssrs.
of particular interest for map merging is the fact that  <dig> ssrs  were polymorphic in two or more mapping populations, suggesting that these common markers may serve as anchoring points across maps.
overall, the percentage of potentially mapable markers in the f <dig> families, as resolved by high-resolution agarose gel electrophoresis, ranged from  <dig> %  to  <dig> % .
codominant segregation was observed in 38%  to 78%  of the segregating markers, whereas dominant segregation accounted for 22%  to 62%  of the segregating markers.
rs - sugar type  in roots
[ ×
rs - sugar type  in roots
p <dig> - purple/yellow pigment accumulation in roots
×
substantial variation in the degree of polymorphism was found between the two sets of markers .
in general, gssrs were more polymorphic than bssrs.
depending on the f <dig> family,  <dig>  - 35%  of the gssrs, and  <dig>  -  <dig> %  of the bssrs, were polymorphic.
overall, nearly 77% of gssrs and 52% of bssrs were polymorphic in at least one f <dig> family.
the mean polymorphism index , which takes in account the type of polymorphism , was significantly higher  for gssrs  compared to bssrs , regardless of the mapping population.
ssr polymorphism -as evaluated in  <dig> mapping populations- was related to repeat number.
figure  <dig> presents mean polymorphism indexes for perfect microsatellites across four repeat number classes.
a general trend showing increasing rates of polymorphism associated with increased repeat number was observed.
supporting this observation, significant correlations  between number of repeat units -and total ssr length- and polymorphism index  were detected.
also, the percentage of polymorphic loci  followed a similar trend across the repeat number classes, with values of  <dig> %,  <dig> %,  <dig> % and  <dig> % for ssr markers with <  <dig>  6- <dig>  11- <dig>  and >  <dig> repeat units, respectively .
markers harboring multiple perfect microsatellites in their amplicon sequence  with 11- <dig> repeat units were the most polymorphic markers, followed by long dinucleotides and tetranucleotides with more than  <dig> repeat units.
with the exception of trinucleotides, for which there was no clear increase in polymorphism with increased repeat number, the ssr markers considered altogether revealed a clear positive relationship between the two variables, with a nearly 3-fold increase in polymorphism when comparing the lowest  and highest  repeat number classes.
marker transferability across apiaceae
a total of  <dig> ssr loci were assessed across  <dig> apiaceae accessions for a total of  <dig>  primer/accession combinations.
of these,  <dig>   produced fragments within the expected size range.
combinations that produced fragments outside the expected size range  were considered non-specific amplifications and regarded as negative results.
this range was arbitrarily selected to simplify the analysis, especially in the cases where more than two bands were amplified.
all successful amplifications were obtained at annealing temperatures between  <dig> and -2° of the recommended value.
alternative pcr protocols, such as touchdown, did not significantly improve amplification success.
the potential transferability of ssrs across apiaceae taxa varied widely among the accessions  and this was highly associated with the accessions' phylogenetic relatedness to carrot, the species from which the markers were developed .
thus, for carrot  accessions, the total number of markers that produced amplicons of expected size was high, and ranged from  <dig> , for the wild carrot 'qal', to  <dig> , in the nantes-type french cultivar 'de la halle', with a mean value of  <dig>  markers.
pcr amplification efficiencies in non-carota daucus accessions were intermediate between carrot and non-daucus apiaceae accessions, with a mean of  <dig>  successful amplifications, and ranging from  <dig>  in d. gutattus to  <dig>  in d. capillifolius.
as expected, d. capillifolius, which is a close relative of d. carota and the only species in our data set with the same chromosome number as carrot , had the highest success rate, almost as high as the d. carota accessions .
the more distantly related non-daucus apiaceae species generated ssr amplicons with  <dig>  to  <dig>  of the markers, with an average of  <dig>  for this group.
again, the species most closely related to carrot within this group, o. grandiflora, had higher ssr amplification efficiency than other non-daucus accessions.
consistent with the previous analysis, the number of ssr primer pairs that produced expected-size amplicons across all the daucus carota accessions , non-carota daucus  and apiaceae  was  <dig>   <dig>  and  <dig>  respectively.
the performance of each ssr marker across the  <dig> apiaceae accessions is presented in additional file  <dig> - table s <dig>
linkage mapping
of the  <dig> ssr markers evaluated for polymorphism and mode of segregation in the b <dig> × qal population ,  <dig>  were monomorphic and  <dig>  polymorphic, whereas  <dig> ssrs  did not produce amplicons and  <dig>  yielded ambiguous band patterns that did not allow their inclusion in the previous classes.
the polymorphic markers were assayed in the entire f <dig> population.
of these,  <dig> ssr markers were omitted because severe segregation distortion and/or multiple pcr products  were observed.
the remaining  <dig> markers - <dig> bssrs and  <dig> gssrs- generated robust and easily interpretable genotypes that could satisfactorily be used for individual genotyping and genetic mapping.
these included  <dig> codominant and  <dig> dominant ssrs, which were successfully placed in the carrot reference linkage maps .
no segregation distortion was detected in this ssr marker array as evaluated by f <dig> chi-square segregation analyses.
the parental maps of qal and b <dig> included  <dig>  and  <dig>  ssrs, respectively.
these include a codominant ssr marker  previously mapped in lg <dig>  the mapped ssrs were distributed across all  <dig> linkage groups  of the carrot genome, with 2- <dig> and 2- <dig> markers/lg in the qal and b <dig> maps, respectively.
only  <dig> and  <dig> map intervals with genetic distance greater than  <dig> cm  scattered throughout different lgs were observed in the b <dig> and qal maps, respectively, indicating a relatively evenly distributed map coverage.
a comparative summary of both parental maps, by linkage groups, is presented in table  <dig>
* a = aflps; t = transposon display markers; g = gene-derived markers; s = scar markers
ǂ mean distance between markers is the arithmetic mean of map distances between adjacent markers shown in figure  <dig>
† arithmetic mean of the mean distance between markers in each linkage group
overall, after mapping the ssr loci, the linkage map of the wild carrot qal contains  <dig> molecular markers  covering  <dig> .
<dig> cm, with an average distance between adjacent markers of  <dig>  cm, whereas the cultivated b <dig> map harbors  <dig> markers  covering  <dig>  cm, with a  <dig>  cm average marker distance.
thus, although the parental b <dig> map includes fewer markers, it has a larger total map length than the qal map.
a paired t-test revealed a significantly higher  mean recombination fraction between adjacent markers  when comparing the two parental maps.
although marginally significant, the higher mean recombination found in b <dig> may help explain its larger observed total map length.
because in a very recent study  <cit>  the linkage groups  from this map were integrated with actual chromosomes by means of flourescent in situ hybridization  mapping of bac clones anchored by lg-specific markers , the lgs in figure  <dig> were named, ordered and oriented north-south according to the corresponding chromosomes.
by convention, chromosomes are numbered consecutively from longest to shortest, and they are oriented with their short and long arms following north-south directions.
thus, correspondences between our lg designations and those from previous maps  <cit>  are as follows: lgs  <dig>   <dig>   <dig>   <dig>  and  <dig> correspond to former lgs  <dig>   <dig>   <dig>   <dig>  and  <dig>  respectively, with conserved orientation, whereas lgs  <dig>   <dig>   <dig>  and  <dig>  in this study  correspond to former lgs  <dig>   <dig>   <dig>  and  <dig>  respectively, with inverted north-south orientations.
the  <dig> fully informative  ssr markers consistently mapped to homologous lg pairs of the cultivated  and wild  carrot genomes .
in addition, for these common markers, complete conservation of locus order was observed along the individual lgs between the two maps .
this was also true for other -previously mapped- codominant markers .
thus, no evidence of rearrangement of chromosomal blocks between the wild and the domesticated carrot subspecies was found.
within each lg, ssrs were frequently positionally associated to genes.
more than 40% of the ssr markers of both maps  mapped within  <dig> cm from previously mapped genes, whereas  <dig> of the  <dig>  carotenoid genes - in both, qal and b <dig> maps- had  <dig> or more ssrs within a  <dig> cm distance, suggesting that these repeats are relatively frequent in genic regions of the carrot genome.
ssr diversity in daucus carota accessions
our diversity analysis including  <dig> cultivated and wild carrots revealed valuable information on the degree of polymorphism of ten selected microsatellite loci.
table  <dig> presents the number of alleles , allele lengths, and expected heterozygosity  found for these ssrs in our d. carota diversity collection.
for this germplasm,  <dig> different alleles, with lengths ranging from  <dig> to  <dig> bp, were identified.
all the loci examined were highly diverse.
the average number of alleles per ssr was  <dig>  with a range of 10- <dig>  whereas the mean expected heterozygosity was  <dig> , and ranged from  <dig>  for gssr <dig> to  <dig>  for gssr <dig>  the most polymorphic loci were gssr <dig>  and gssr <dig> , and the least polymorphic was gssr <dig> .
discussion
frequency and distribution of ssrs in carrot genomic and est sequence
microsatellite density in genomic dna of carrot was estimated by analysis of  <dig>  mbp of bac end sequence .
carrot had a rather low ssr density  compared to other species.
ssrs analyses -using the same search parameters and program as with carrot- in the complete genome sequences of four model species revealed ssr densities of  <dig>   <dig>   <dig>  and  <dig> ssrs/mbp in arabidopsis thaliana, grapevine, rice, and poplar, respectively.
the lower ssr density in carrot compared to these species cannot be attributed to differences in the source of genomic sequence  since analyses of bac end sequence  datasets from these and other species were also much more dense in microsatellites than carrot bes .
similarly, transcript sequences of carrot , although more dense in ssrs than their genomic counterparts, were also less frequent in these repeats compared to ests of arabidopsis , grapevine , poplar , soybean , rice , and sorghum .
carrot trinucleotides were more frequent in transcripts than in genomic dna.
in addition, within bssrs trinucleotide repeats occurred preferentially inside orfs, and accounted for ~ 50% of the total ssrs found in these protein coding regions.
the abundance of these repeats in ests and in orfs is consistent with the notion that protein-coding sequences tolerate better frame-shift mutations  of  <dig> bp -or multiples of  <dig> bp- than other indel lengths.
thus, trinucleotide repeats within coding sequences may translate fully functional proteins with a few extra  aminoacids, whereas indels of other lengths would translate abnormal, often deleterious, proteins.
consistent with our results, an overrepresentation of trinucleotides in protein-coding sequences has been reported previously in numerous plant species  <cit> , as well as in other eukaryotes including humans, primates, rodents and insects  <cit> .
the relative abundance of trinucleotides over other ssr types has been attributed not only to negative selection against frame-shift mutations in the coding regions but also to positive selection for specific single amino-acid stretches  <cit> .
dna polymerase slippage is the main mutational mechanism leading to changes in microsatellite length  <cit> .
these changes in ssr size are most often gradual and step-wise since polymerase slippage only generates gains or losses of one or a few repeat unit  <cit> .
thus, the fact that ssrs in carrot transcripts generally had fewer repeat units than ssrs in genomic sequence, even for trinucleotide repeats , suggests a negative selection pressure against microsatellite size increase in protein-coding sequences.
the non-random distributions of motif sequences among dinucleotide and trinucleotide ssrs of carrot included a higher than expected incidence of n repeats in genomic dna , like that of several plant species including soybean, arabidopsis and rice  <cit> , but unlike the n predominant motif among dinucleotides in humans  <cit> .
in contrast, the n motif was less often observed in ests than expected, while n and n were more common than expected.
this may suggest different constraints for repeat motifs across diverse organisms.
marker development and analyses in f <dig> families
in this study, two different strategies were used for isolating and developing carrot ssr markers.
the hybridization-based approach, as described by glenn and schable  <cit> , yielded microsatellites  that were, in average, significantly longer  and had more repeat units  than ssrs from bac end sequences .
these differences are, most likely, due to differences in the two strategies used.
dna library enrichment methods based on hybridization capture  <cit>  are generally designed to yield a higher proportion of ssrs with large number of repeat units, targeting mainly long perfect repeats.
under this system, long dna stretches of perfect repeats are hybridized more efficiently to the microsatellite probes and they are retained at a higher rate, compared to short repeats, during the washing steps, thus, increasing the relative proportion of long microsatellite sequences in cloned colonies  <cit> .
conversely, the bssrs set represents a random sample -without enrichment for length, repeat type or sequence motif- from genomic dna.
because of this, they provide a more reliable picture of the microsatellite distribution in the carrot genome.
longer and more repetitive ssrs have been obtained through hybridization-based methods compared to sequence-searches in other plant species, regardless of the type of dna examined , including brassica  <cit> , cotton  <cit> , wheat and rice  <cit> .
the differences in repeat number and length between gssrs and bssrs have important implications for marker potentiality, particularly with regard to polymorphism.
in general, gssrs were significantly more polymorphic than bssrs, considering both the polymorphism index   and the percentage of polymorphic markers , and these differences were associated to a higher repeat number and length in the gssrs group .
developments of ssr markers from other plant species, including cotton  <cit> , barley  <cit>  and pine  <cit> , have also noted positive relationships between ssr polymorphism and number of repeat units.
together, these results are consistent with studies reporting that both ssr polymorphism and ssr mutation rate have a positive relationship with repeat number  <cit> .
concordantly, positive and significant relationships have also been found between repeat length and mutation rate in human  <cit> , fruit fly  <cit>  and yeast  <cit>  microsatellites.
these studies indicate that polymerase slippage, the main mutational mechanism in microsatellites  <cit> , increases with higher repeat number and length, leading to a higher diversity in longer, more repetitive ssrs, as observed in the present study.
however, contrary to these and our results, studies using markers developed from other plants, such as brassica  <cit>  and pearl millet  <cit> , have reported lack of correlation between size of the ssr, both measured by length  and repeat number, and detection of polymorphic loci.
as pointed out in the latter two studies, ssr evolutionary age is a key factor for ssr diversity  and this may help explain the lack of association found by them.
in addition, most of the above studies  cannot rule out the possibility that indels at regions other than the ssr motifs may account for some of the polymorphisms, thus influencing the expected relationship between length and polymorphism.
a major interest for evaluating the ssr markers in the carrot f <dig> populations was to assess their potential for mapping.
linkage maps using some of these f2s have already been constructed  and others are underway .
these maps include different phenotypic traits of interest  and -before this study- they were mainly constructed using anonymous dominant markers, such as aflps and rapds, with only very few markers, or none, in common, thus, making their comparative analyses and/or integration difficult.
the present work identified  <dig> ssrs  that were polymorphic in two or more mapping populations, suggesting that these common markers may serve as anchoring points for merging carrot maps.
besides the inclusion of  <dig> ssr markers onto the carrot reference map , work is underway in our lab to include these polymorphic ssrs in other maps with different genetic backgrounds .
the integration of carrot linkage maps would enhance their usefulness for assisting breeding of this species, by increasing marker saturation nearby genes of interest and thereby facilitating applications like positional gene cloning, among others.
from our evaluation in seven carrot f <dig> families,  <dig> ssr markers  were polymorphic in at least one mapping population.
because the pcr amplicons were size-separated using high-resolution agarose gel electrophoresis, which can only resolve fragments with size differences of at least  <dig> bp, a fraction of the markers evaluated in some populations, generated ambiguous band patterns.
although they may have been polymorphic, the bands were too close together in the gel to unambiguously score, and were classified as monomorphic .
thus, if other fragment separation systems, with better resolution, are used, such as separation of fluorescently-labeled fragments through capillary electrophoresis, the number of polymorphic markers may be expanded in some populations, particularly in cases of dinucleotide ssr markers varying in a single repeat unit.
high pcr amplification efficiencies were found in the f <dig> families for both sets of markers, gssrs  and bssrs .
comparable amplification efficiencies have been found in other plant species with ssr markers developed using hybridizations-based methods  and sequence-based searches .
transfer success of carrot ssrs across apiaceae
the availability of ssr loci for economically important species has increased interest in primer transferability to related taxa, especially for species in which molecular resources are limited.
in apiaceae, only a few publicly available ssrs have been reported previously, and these were developed from carrot  and celery , the two most economically important crop species in the family.
results from this study indicate that a significant fraction of carrot ssrs transfer successfully across apiaceae.
locus amplification success was detected in  <dig> to  <dig> markers across  <dig> non-carrot apiaceae species, including economically important crops like parsley , celery  and cilantro .
prospects of a broader utilization of these markers beyond carrot include their application in taxonomic, population, and conservation studies as well as for mapping and assisting breeding in crop species.
it is, however, important to bear in mind that when using ssr markers across distantly-related species the amplification of a pcr product does not necessarily imply locus conservation, since size homoplasy, i.e.
convergence in size of non-homologous fragments, may occur.
considering the possibility of this source of confusion, verification of the pcr product identity by sequencing has been suggested previously, particularly when working across genera and if there is uncertainty regarding the size range of the amplicons obtained  <cit> .
however, verification through sequencing may not be necessary if working within the same genus as the species from which the ssrs markers were developed  <cit> .
thus, the use of carrot ssr markers for studies in non-daucus apiaceae should include verification, by sequencing, of the homology to the carrot ssr product sequence .
transfer of carrot ssrs across daucus species  was, in general, less successful than ssr transfer rate at the subgenus level reported for other species, whereas transfer of carrot ssrs across-genera was relatively higher than found in other plants.
according to a previous review of ssr cross-transferability in plants  <cit> , the average transferability across species in the same genus was  <dig> %, and across related genera was  <dig> %.
we found these values to be  <dig> % across daucus species and 41% across the apiaceae.
however, it should be noted that ssr transfer success varied greatly across the different reports for both within-same genus  and across different genera   <cit> .
the huge variation found across these studies likely reflects differences in phylogenetic distance  between the source and target taxa within each family, as well as differences in the number of taxa and ssr loci analyzed, and in the type of sequences used for marker development.
for example, est-derived ssrs are more conserved and thus they transfer across genera more readily than genomic ssrs  <cit> ), among other factors.
our data  suggest generally a higher rate of success in amplifying carrot ssrs in plants more closely related to carrot.
this should not be surprising since closer-related taxa have higher overall sequence homology which translates to more conserved ssr flanking regions and, therefore, easier transferability of primer pairs.
negative relationships between ssr transfer success and phylogenetic distance between source and target taxa have been widely observed in many plant families  <cit> .
the potential usefulness of ssr markers for diversity and phylogenetic studies in apiaceae will depend, to a great extent, on the possibility that markers successfully amplify across different species and on the ability of the marker to detect polymorphism among the taxa.
to have a preliminary picture of how suitable the ssr markers developed in this work may be for these applications, we investigated interspecific ssr variation among non-carrot species by analysis of amplicons sizes in the agarose gel images.
thus, for each ssr, the total number of different alleles in the non-carrot species dataset was recorded .
only ssrs that successfully amplified products in at least 80% of the non-carrot species  were considered.
overall, our results revealed  <dig> ssrs that generated amplicons in most  outside-carrot species.
of these,  <dig> ssrs  produced 3- <dig> different alleles  in the non-carrot group.
it should be noted that our calculation of  <dig>  alleles/ssr in these selected markers is conservative, due to the low resolution of agarose gels which do not allow discrimination of different alleles varying in one or a few repeats.
these results suggest that a significant proportion of the ssr markers developed herein may be suitable for addressing taxonomic or phylogenetic questions within apiaceae.
further analysis of the  <dig> ssrs that produced amplicons in the majority of the non-carrot taxa revealed interesting differences between the two ssr datasets.
although more bssrs than gssrs  amplified successfully in most non-carrot taxa, gssrs were much more polymorphic than bssrs at the interspecific level.
for example, among gssrs  <dig> markers produced  <dig> or more different alleles , whereas only  <dig> bssrs generated  <dig> or more alleles/ssr .
it is likely that the generally higher polymorphism of gssrs compared to bssrs at the inter-specific level, which is in agreement with our results for both sets of markers in the carrot f2s, may be also due to the higher number of repeat units present in gssrs.
ssr linkage mapping
prior to this work, important advances were made in the construction of carrot genetic maps with a range of molecular marker systems.
although some rflps  <cit>  and a few scar and gene-specific markers were mapped  <cit> , the most extensive genetic mapping data in carrot has been generated mainly with dominant aflp, rapd and transposon-display  markers  <cit> .
while rflps are useful for comparative mapping purposes, high throughput genotyping and probe handling are difficult.
similarly, the carotenoid genes mapped by just et al.
<cit>  are not as easily transferred to other mapping backgrounds since their analysis relied in most cases on snps, due to the lack of larger polymorphisms  in these genes that can be scored as easily as ssrs.
on the other hand, aflp, rapd and td markers, while providing a relatively large number of markers per assay and good genome coverage, have limited information content and are not of much use for comparative mapping purposes and for validating qtl across pedigrees  <cit> .
the addition of  <dig> ssr markers to the carrot reference linkage map together with detailed characterization of this novel set of  <dig> ssrs in subsets of six other mapping populations should allow significant advances in carrot comparative mapping and map-integration.
the fact that most of the mapped ssrs were codominant  in the b <dig> × qal-derived population, with 2- <dig> informative markers per linkage group, together with the identification of putative codominant ssrs in other mapping populations adds extra value to the data published here for pursuing these goals.
the inclusion of ssrs in linkage maps with additional pedigrees is currently underway.
the parental b <dig> map has a slightly larger total map length than the qal map.
although the higher mean recombination found in b <dig> may help explain its larger map length, other factors -e.g., related to the type of markers used- may also cause this effect.
different recombination frequencies can be obtained just by sampling of the different markers, as well as errors derived from calculations of genetic distances from dominant markers data.
in the current map, we have modified linkage group designations and orientations, in accordance to recent cytogenetic data concerning the integration of carrot lgs with actual chromosomes  <cit> .
following standard conventions, consecutive numbers were assigned to the lgs in decreasing order of chromosome length , and four lgs were inverted in their north-south orientations to agree with the standard short arm/long arm presentation of their corresponding chromosomes.
it must, however, be noted that although all the lgs could be unequivocally associated to chromosomes, and thus their number designations are correct and complete, unambiguous lg orientations could only be defined for six of the nine lgs.
thus, chromosomes  <dig>   <dig>  and  <dig> in the current map, which correspond to former lgs  <dig>   <dig>  and  <dig>  respectively, could not be unequivocally oriented, because a single anchored bac probe was used for lg-chromosome integrations.
thus, their orientations were not modified from previous map versions  <cit> .
however, the possibility remains that future cytogenetic data  may reveal different orientations for these lgs.
these modifications based on recent cytogenetic data, and the addition of  <dig> new ssr markers, add value to the updated reference carrot linkage map presented herein.
overall, the current maps involve 193- <dig> mapped loci, including  <dig> highly informative markers which consist of ssr, carotenoid gene and scar markers, spanning  <dig> - <dig>  cm, making it the most comprehensive genetic linkage map in the apiaceae to date.
the ssr loci mapped across all  <dig> lgs in both parental maps, and they were distributed fairly evenly within most individual lgs, thus recommending their usefulness as anchor points for merging carrot maps.
in addition, such dispersed map distribution of the ssr loci, has allowed us to develop bac fish probes carrying ssr sequences mapped to specific lgs.
these ssr-anchored probes were used for integrating some lgs of carrot with chromosomes by fish mapping  <cit> .
the positional association observed between ssrs and previously mapped genes suggests that these tandem repeats are frequent in genic regions of the genome.
this is in agreement with results of morgante et al.
<cit> , demonstrating higher microsatellite frequencies in the transcribed and non-repetitive fractions of plant genomes.
one ssr  and two ssrs  in lg <dig> and lg <dig>  respectively, were located in the vicinity of two highly-significant quantitative trait loci  for total root carotene accumulation.
these correspond to the y and y <dig> loci, respectively, described by buishand and gabelman  <cit> .
microsatellites gssr <dig> and gssr <dig>  although not tightly linked to y <dig>  may be useful for marker assisted selection, either as a complement of, or as an alternative to the lack of amplification of other robust more-closely linked markers, such as y2mark  <cit> .
CONCLUSIONS
this work reports on the development and characterization at various levels of a novel set of  <dig> carrot ssr markers.
analysis of the distribution of ssr motifs, repeat lengths and polymorphism across genomic and est sequences, as well as concerning different ssr isolation methods  may help decide over future strategies for developing valuable ssr markers in this and other species.
the genetic mapping of  <dig> ssr loci onto the reference carrot linkage map, distributed throughout all  <dig> linkage groups, together with the characterization of the entire set of markers in  <dig> other mapping populations, should facilitate comparative mapping studies and integration of carrot maps.
particularly important for these purposes are the  <dig> codominant ssrs added to the map, which resulted in more than doubling the original number of informative markers in this -or any other- carrot map reported to date.
in addition, ssr evaluations in carrot-related taxa indicates that a significant fraction of the carrot ssrs transfer successfully across apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot.
nonetheless, a fairly large number of potentially useful ssr markers were identified for non-carrot daucus  and non-daucus apiaceae  species, increasing the prospects of their successful utilization in other apiaceae.
in addition, allelic diversity at selected ssr loci was evaluated using  <dig> d. carota accessions.
in this germplasm, the microsatellites proved to be highly polymorphic, with an average of  <dig> alleles/locus and  <dig>  expected heterozygosity.
the marker resources developed in this work should be a valuable tool for carrot breeding and genetics.

 the apiaceae family contains a number of important vegetable and spice crop species including carrot, celery, fennel, cilantro, parsley and parsnip. carrot is the most economically important species in the apiaceae, with more than million tons produced yearly worldwide. carrot consumption has steadily increased in recent decades for several reasons, including a heightened awareness of its health-promoting attributes, development of fresh-cut carrot products convenient for consumers, and adaptation of improved cultivars for warmer production areas. despite their economic importance, carrot and other apiaceae have relatively underdeveloped molecular resources. three unsaturated linkage maps, mainly based on anonymous dominant aflp markers, have been constructed in carrot to assist breeding of this species. inheritance studies on natural carotenoid mutants have identified factors conditioning root pigment accumulation, and both simply inherited pigment traits and qtl have been included in these maps. the more recent addition of genes from the carotenoid biosynthetic pathway, as well as transposon-display markers, onto one of the maps has increased both maker informativeness and coverage. the latter map, which was constructed using an f family derived from a cultivated × wild carrot cross, is considered the carrot reference map because it harbors important phenotypic traits, has fairly good coverage and includes the largest number of informative markers. however, direct comparisons between the reference and other carrot maps are currently difficult due to the lack of common markers across maps. this fact seriously limits the usefulness of these maps for assisting breeding, especially considering that a number of important carrot traits, including nematode resistance, anthocyanin pigmentation and reduced-sugar accumulation, have been mapped in unrelated genetic backgrounds. the lack of common markers across carrot maps is mainly due to an insufficient availability of informative and robust pcr-based markers. the carotenoid genes mapped by just et al. are not easily transferred to other maps with different genetic backgrounds, since they were mainly mapped by sequence detection of single polymorphism nucleotides, rather than by fragment length polymorphisms, and because sequence conservation was very high in some carotenoid genes. thus, for these genes, little snp polymorphism may be expected. the development of polymorphic and robust pcr-based markers in carrot, such as microsatellites, would facilitate their inclusion in different maps, thus serving as anchoring points for map integration. this would immediately increase map marker density, ssr-tagging relevant phenotypic traits and, perhaps, facilitate applications such as positional gene-cloning. in addition, other carrot genetic research studies, such as analysis of genetic diversity and phylogenetic reconstructions, previously approached using anonymous dominant aflp markers or laborious time-consuming codominant rflps, would also benefit from the development of microsatellite markers. microsatellites, or simple sequence repeats, are the marker of choice in many molecular genetic applications including mapping, fingerprinting, genetic diversity, population structure analysis, gene flow and germplasm conservation studies. their widespread adoption is due to several desirable characteristics: they are codominant, frequently and evenly distributed throughout genomes, selectively neutral, highly reproducible and rely on simple polymerase chain reaction technology. in addition they are ubiquitous and hypervariable. the latter property is attributed to a high mutation rate of these repeats resulting from dna polymerase slippage during dna synthesis. this mutational mechanism generates gains or losses of one or a few repeat units in the microsatellite, which accumulate more rapidly than point mutations and indels, leading to a high number of alleles per locus. in plants, the high polymorphism found in microsatellites has allowed the detection of variability in species otherwise characterized by low levels of genetic diversity. in apiaceae, very few publicly available ssrs have been reported previously, and these were developed from carrot and celery, the two most economically important species in the family. the availability of a large set of ssrs in carrot is likely to benefit research in other apiaceae, since significant marker transferability has been observed across related taxa. this is of particular interest to research groups working in minor crops or species with limited research funds; many laboratories have sufficient resources and expertise for running ssr-based pcr analyses, although perhaps not for the isolation and characterization of new loci. in this study we report on the development of new carrot ssr markers. further characterization of these loci includes analysis of ssr distributions in genomic and est sequence, linkage mapping onto the carrot reference map, evaluation of their mapping potential in subsets of seven carrot f mapping populations, evaluation of their potential for assessing genetic diversity among daucus carota accessions, and evaluation of ssr marker transferability across apiaceae taxa.  distribution of microsatellites in genomic and est sequence substantial variation in ssr frequency and distribution between genomic and est sequence of carrot was found. because frequency distributions for gssrs mainly reflect the library enrichment procedure, conditioning the type, length and sequence motifs of the ssrs obtained, only ssrs from bac end sequence were used as representatives of genomic ssrs in comparisons with ssrs from est sequence. overall, ssrs occurred at a lower density in genomic dna than in ests. dinucleotide, trinucleotide, and tetranucleotide repeats predominated in genomic dna, whereas in ests trinucleotides were the predominant repeats accounting for 50% of the total ssrs. the absolute density for this repeat type in est data was nearly twice its density found in genomic sequence. the same was observed for dinucleotide and hexanucleotide repeats, which were more than twice as frequent in ests compared to genomic dna. conversely, the density of pentanucleotides, heptanucleotides and octanucleotides was more than fold higher in genomic sequence than in est sequence. * a minimum of repeat units for dinucleotides, r.u. for trinucleotides, and r.u. for tetra-, penta-, hexa- hepta- and octanucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot. ǂ only density values for ssrs from bac end sequences and from ests are presented. ‡ ssr motif considering complementary; ssr motifs occurring at a rate of at least 5% of the total are listed with occurrence as percentage within each ssr type class in parentheses; ssr classes without predominance of a particular motif are denoted as "-". ζ total length of sequences analyzed. γ includes both gssrs and bssrs. comparisons among microsatellites isolated from the different sequence datasets revealed that di-, tri-, and tetranucleotide repeats accounted for % to % of the ssrs among the three types evaluated, with di- and tetranucleotide motifs accounting for most of the gssrs, while trinucleotide motifs were most common among bssrs and essrs. within genomic dna, variation was also found between the two sequence datasets examined. overall, gssrs had more repeat units and consequently were longer than bssrs. the larger number of repeat units in gssrs compared to bssrs was evident and significant for di, tri, and tetranucleotides, whereas penta to octanucleotides had the same mean number of repeats in both datasets. ssr length had a similar relationship for these repeat types in both datasets. with regard to repeat types, gssrs yielded a higher proportion of di and tetranucleotides, as compared to bssrs, whereas trinucleotides and penta to octanucleotides were more frequent in the latter group. in addition, gssrs included a significant fraction of often long, compound microsatellites, such repeats being nearly times more frequent in this group compared to bssrs. based upon posterior probabilities, the distributions of sequence motifs n, n, n, etc.; n, n, n, etc.) in the dinucleotide and trinucleotide classes were not random for bssrs and essrs. in bac end sequence, n and n dinucleotides were more and less frequent than expected, respectively. in ests, n repeats were less frequent than expected whereas n and n dinucleotides occurred at a higher-than-expected incidence. ag and ac motifs occurred frequently in dinucleotide ssrs of all sequence origins, although at dinucleotides were most frequent among bssrs. repeats of aag, act, and aaat were abundant and common to both genomic and essrs. on the other hand, repeats of aac, agt, acat, aatt, and aaaag, predominated mainly among genomic microsatellites, whereas agg, agc, aaag, agccc, aaaaag, and aaagag motifs were most frequent in est ssrs. comparisons between observed and expected trinucleotide repeat motifs presented no clear trends but observed distributions differed from those expected for many motifs to result in significant deviation based upon chi-square analysis. *a minimum of repeat units for dinucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot. chi-squared distributions indicate comparison of observed ssr distribution of ssr motif types n, n, n, etc.; n, n, n, etc.) to posterior probabilities. tests are partitioned by library and sequence motif. χ = chi-square statistic; df = degrees of freedom. ssrs were non-randomly distributed across motif types. microsatellite distribution was not uniform across coding and non-coding regions of carrot. frequency distributions of both repeat types and sequence motifs for each microsatellite origin, i.e., a library enrichment procedure, bac end-derived and est-derived ssrs, varied markedly across these dna fractions. among gssrs, di- and tetranucleotide repeats were most common, and tetranucleotide repeats were distinctive in being the only gssr repeat type with a significantly different occurrence inside and outside of orfs, with over 70% of tetranucleotide ssrs inside orfs. analysis of repeats in bac end sequence revealed a predominance of trinucleotides in coding sequences, compared to non coding regions, whereas tetranucleotides were somewhat more abundant in non-coding regions. the overrepresentation of trinucleotides in orfs of genomic dna was higher than expected by chi-square analysis and associated to a high frequency of aag, aac, agt, act, acg, and acc motifs, whereas non-coding regions rich in tetranucleotides were particularly gc-poor with an abundance of aaat and aatt motifs. dinucleotide, pentanucleotide, and hexanucleotide repeats were nearly equally frequent in protein coding and non-coding regions of bac end sequence. * a minimum of repeat units for dinucleotides, r.u. for trinucleotides, and r.u. for tetra-, penta-, hexa-, hepta-,and octanucleotides were used as parameters for searching microsatellites in genomic and est sequence of carrot. chi-square distributions indicate observed ssr motif types inside versus outside orfs as compared to posterior probabilities. χ = chi-squared statistic; df = degrees of freedom. observed distributions generally fit those expected except for tetranucleotides for gssrs, and trinucleotides for bssrs. for essrs the observed distributions only fit expected ones for tri-, penta-, and hexanucleotide repeats, with observed occurrence of di- and tetranucleotide repeats occurring much more often outside orfs than expected. the distribution of ssr types in est sequences was especially variable, with trinucleotides predominating in orfs, representing more than 65% of the ssrs found inside orfs and 26% of all essrs, and dinucleotides and tetranucleotides predominating in non-protein coding sequences of the ests. in est orfs, the most frequent trinucleotide motifs were aag, act, agg, agc, and acc. in the dinucleotides-rich utr region of ests, ag and ac motifs were three times more frequent than in protein-coding regions of ests. ssr marker development primer pairs for gssr and bssr loci were designed. of these primers pairs, flanked single ssrs and flanked multiple ssrs. markers with single perfect repeats included dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, hexanucleotides, heptanucleotides, and each of mono, octa and nonanucleotides. further information on these ssr markers is presented in additional file - table s including primer sequence, annealing temperature, repeat motif and its position in template sequence, expected amplicon length, and the template dna sequence carrying the ssr. for a number of microsatellites detected computationally, we were not able to design primers because they either lacked suitable flanking sequences or the total sequence length was too short. marker polymorphism analyses in carrot f families a total of ssrs were successfully characterized in subsets of carrot f families. details on the performance of each ssr marker in the different mapping populations are presented in additional file - table s overall, ssrs were polymorphic in at least one mapping population. these included gssrs and bssrs. of particular interest for map merging is the fact that ssrs were polymorphic in two or more mapping populations, suggesting that these common markers may serve as anchoring points across maps. overall, the percentage of potentially mapable markers in the f families, as resolved by high-resolution agarose gel electrophoresis, ranged from % to %. codominant segregation was observed in 38% to 78% of the segregating markers, whereas dominant segregation accounted for 22% to 62% of the segregating markers. rs - sugar type in roots [ × rs - sugar type in roots p - purple/yellow pigment accumulation in roots × substantial variation in the degree of polymorphism was found between the two sets of markers. in general, gssrs were more polymorphic than bssrs. depending on the f family, - 35% of the gssrs, and - % of the bssrs, were polymorphic. overall, nearly 77% of gssrs and 52% of bssrs were polymorphic in at least one f family. the mean polymorphism index, which takes in account the type of polymorphism, was significantly higher for gssrs compared to bssrs, regardless of the mapping population. ssr polymorphism -as evaluated in mapping populations- was related to repeat number. figure presents mean polymorphism indexes for perfect microsatellites across four repeat number classes. a general trend showing increasing rates of polymorphism associated with increased repeat number was observed. supporting this observation, significant correlations between number of repeat units -and total ssr length- and polymorphism index were detected. also, the percentage of polymorphic loci followed a similar trend across the repeat number classes, with values of %, %, % and % for ssr markers with < 6- 11- and > repeat units, respectively. markers harboring multiple perfect microsatellites in their amplicon sequence with 11- repeat units were the most polymorphic markers, followed by long dinucleotides and tetranucleotides with more than repeat units. with the exception of trinucleotides, for which there was no clear increase in polymorphism with increased repeat number, the ssr markers considered altogether revealed a clear positive relationship between the two variables, with a nearly 3-fold increase in polymorphism when comparing the lowest and highest repeat number classes. marker transferability across apiaceae a total of ssr loci were assessed across apiaceae accessions for a total of primer/accession combinations. of these, produced fragments within the expected size range. combinations that produced fragments outside the expected size range were considered non-specific amplifications and regarded as negative results. this range was arbitrarily selected to simplify the analysis, especially in the cases where more than two bands were amplified. all successful amplifications were obtained at annealing temperatures between and -2° of the recommended value. alternative pcr protocols, such as touchdown, did not significantly improve amplification success. the potential transferability of ssrs across apiaceae taxa varied widely among the accessions and this was highly associated with the accessions' phylogenetic relatedness to carrot, the species from which the markers were developed. thus, for carrot accessions, the total number of markers that produced amplicons of expected size was high, and ranged from, for the wild carrot'qal', to, in the nantes-type french cultivar'de la halle', with a mean value of markers. pcr amplification efficiencies in non-carota daucus accessions were intermediate between carrot and non-daucus apiaceae accessions, with a mean of successful amplifications, and ranging from in d. gutattus to in d. capillifolius. as expected, d. capillifolius, which is a close relative of d. carota and the only species in our data set with the same chromosome number as carrot, had the highest success rate, almost as high as the d. carota accessions. the more distantly related non-daucus apiaceae species generated ssr amplicons with to of the markers, with an average of for this group. again, the species most closely related to carrot within this group, o. grandiflora, had higher ssr amplification efficiency than other non-daucus accessions. consistent with the previous analysis, the number of ssr primer pairs that produced expected-size amplicons across all the daucus carota accessions, non-carota daucus and apiaceae was and respectively. the performance of each ssr marker across the apiaceae accessions is presented in additional file - table s linkage mapping of the ssr markers evaluated for polymorphism and mode of segregation in the b × qal population, were monomorphic and polymorphic, whereas ssrs did not produce amplicons and yielded ambiguous band patterns that did not allow their inclusion in the previous classes. the polymorphic markers were assayed in the entire f population. of these, ssr markers were omitted because severe segregation distortion and/or multiple pcr products were observed. the remaining markers - bssrs and gssrs- generated robust and easily interpretable genotypes that could satisfactorily be used for individual genotyping and genetic mapping. these included codominant and dominant ssrs, which were successfully placed in the carrot reference linkage maps. no segregation distortion was detected in this ssr marker array as evaluated by f chi-square segregation analyses. the parental maps of qal and b included and ssrs, respectively. these include a codominant ssr marker previously mapped in lg the mapped ssrs were distributed across all linkage groups of the carrot genome, with 2- and 2- markers/lg in the qal and b maps, respectively. only and map intervals with genetic distance greater than cm scattered throughout different lgs were observed in the b and qal maps, respectively, indicating a relatively evenly distributed map coverage. a comparative summary of both parental maps, by linkage groups, is presented in table * a = aflps; t = transposon display markers; g = gene-derived markers; s = scar markers ǂ mean distance between markers is the arithmetic mean of map distances between adjacent markers shown in figure † arithmetic mean of the mean distance between markers in each linkage group overall, after mapping the ssr loci, the linkage map of the wild carrot qal contains molecular markers covering. cm, with an average distance between adjacent markers of cm, whereas the cultivated b map harbors markers covering cm, with a cm average marker distance. thus, although the parental b map includes fewer markers, it has a larger total map length than the qal map. a paired t-test revealed a significantly higher mean recombination fraction between adjacent markers when comparing the two parental maps. although marginally significant, the higher mean recombination found in b may help explain its larger observed total map length. because in a very recent study the linkage groups from this map were integrated with actual chromosomes by means of flourescent in situ hybridization mapping of bac clones anchored by lg-specific markers, the lgs in figure were named, ordered and oriented north-south according to the corresponding chromosomes. by convention, chromosomes are numbered consecutively from longest to shortest, and they are oriented with their short and long arms following north-south directions. thus, correspondences between our lg designations and those from previous maps are as follows: lgs and correspond to former lgs and respectively, with conserved orientation, whereas lgs and in this study correspond to former lgs and respectively, with inverted north-south orientations. the fully informative ssr markers consistently mapped to homologous lg pairs of the cultivated and wild carrot genomes. in addition, for these common markers, complete conservation of locus order was observed along the individual lgs between the two maps. this was also true for other -previously mapped- codominant markers. thus, no evidence of rearrangement of chromosomal blocks between the wild and the domesticated carrot subspecies was found. within each lg, ssrs were frequently positionally associated to genes. more than 40% of the ssr markers of both maps mapped within cm from previously mapped genes, whereas of the carotenoid genes - in both, qal and b maps- had or more ssrs within a cm distance, suggesting that these repeats are relatively frequent in genic regions of the carrot genome. ssr diversity in daucus carota accessions our diversity analysis including cultivated and wild carrots revealed valuable information on the degree of polymorphism of ten selected microsatellite loci. table presents the number of alleles, allele lengths, and expected heterozygosity found for these ssrs in our d. carota diversity collection. for this germplasm, different alleles, with lengths ranging from to bp, were identified. all the loci examined were highly diverse. the average number of alleles per ssr was with a range of 10- whereas the mean expected heterozygosity was, and ranged from for gssr to for gssr the most polymorphic loci were gssr and gssr, and the least polymorphic was gssr. discussion frequency and distribution of ssrs in carrot genomic and est sequence microsatellite density in genomic dna of carrot was estimated by analysis of mbp of bac end sequence. carrot had a rather low ssr density compared to other species. ssrs analyses -using the same search parameters and program as with carrot- in the complete genome sequences of four model species revealed ssr densities of and ssrs/mbp in arabidopsis thaliana, grapevine, rice, and poplar, respectively. the lower ssr density in carrot compared to these species cannot be attributed to differences in the source of genomic sequence since analyses of bac end sequence datasets from these and other species were also much more dense in microsatellites than carrot bes. similarly, transcript sequences of carrot, although more dense in ssrs than their genomic counterparts, were also less frequent in these repeats compared to ests of arabidopsis, grapevine, poplar, soybean, rice, and sorghum. carrot trinucleotides were more frequent in transcripts than in genomic dna. in addition, within bssrs trinucleotide repeats occurred preferentially inside orfs, and accounted for ~ 50% of the total ssrs found in these protein coding regions. the abundance of these repeats in ests and in orfs is consistent with the notion that protein-coding sequences tolerate better frame-shift mutations of bp -or multiples of bp- than other indel lengths. thus, trinucleotide repeats within coding sequences may translate fully functional proteins with a few extra aminoacids, whereas indels of other lengths would translate abnormal, often deleterious, proteins. consistent with our results, an overrepresentation of trinucleotides in protein-coding sequences has been reported previously in numerous plant species, as well as in other eukaryotes including humans, primates, rodents and insects. the relative abundance of trinucleotides over other ssr types has been attributed not only to negative selection against frame-shift mutations in the coding regions but also to positive selection for specific single amino-acid stretches. dna polymerase slippage is the main mutational mechanism leading to changes in microsatellite length. these changes in ssr size are most often gradual and step-wise since polymerase slippage only generates gains or losses of one or a few repeat unit. thus, the fact that ssrs in carrot transcripts generally had fewer repeat units than ssrs in genomic sequence, even for trinucleotide repeats, suggests a negative selection pressure against microsatellite size increase in protein-coding sequences. the non-random distributions of motif sequences among dinucleotide and trinucleotide ssrs of carrot included a higher than expected incidence of n repeats in genomic dna, like that of several plant species including soybean, arabidopsis and rice, but unlike the n predominant motif among dinucleotides in humans. in contrast, the n motif was less often observed in ests than expected, while n and n were more common than expected. this may suggest different constraints for repeat motifs across diverse organisms. marker development and analyses in f families in this study, two different strategies were used for isolating and developing carrot ssr markers. the hybridization-based approach, as described by glenn and schable, yielded microsatellites that were, in average, significantly longer and had more repeat units than ssrs from bac end sequences. these differences are, most likely, due to differences in the two strategies used. dna library enrichment methods based on hybridization capture are generally designed to yield a higher proportion of ssrs with large number of repeat units, targeting mainly long perfect repeats. under this system, long dna stretches of perfect repeats are hybridized more efficiently to the microsatellite probes and they are retained at a higher rate, compared to short repeats, during the washing steps, thus, increasing the relative proportion of long microsatellite sequences in cloned colonies. conversely, the bssrs set represents a random sample -without enrichment for length, repeat type or sequence motif- from genomic dna. because of this, they provide a more reliable picture of the microsatellite distribution in the carrot genome. longer and more repetitive ssrs have been obtained through hybridization-based methods compared to sequence-searches in other plant species, regardless of the type of dna examined, including brassica, cotton, wheat and rice. the differences in repeat number and length between gssrs and bssrs have important implications for marker potentiality, particularly with regard to polymorphism. in general, gssrs were significantly more polymorphic than bssrs, considering both the polymorphism index and the percentage of polymorphic markers, and these differences were associated to a higher repeat number and length in the gssrs group. developments of ssr markers from other plant species, including cotton, barley and pine, have also noted positive relationships between ssr polymorphism and number of repeat units. together, these results are consistent with studies reporting that both ssr polymorphism and ssr mutation rate have a positive relationship with repeat number. concordantly, positive and significant relationships have also been found between repeat length and mutation rate in human, fruit fly and yeast microsatellites. these studies indicate that polymerase slippage, the main mutational mechanism in microsatellites, increases with higher repeat number and length, leading to a higher diversity in longer, more repetitive ssrs, as observed in the present study. however, contrary to these and our results, studies using markers developed from other plants, such as brassica and pearl millet, have reported lack of correlation between size of the ssr, both measured by length and repeat number, and detection of polymorphic loci. as pointed out in the latter two studies, ssr evolutionary age is a key factor for ssr diversity and this may help explain the lack of association found by them. in addition, most of the above studies cannot rule out the possibility that indels at regions other than the ssr motifs may account for some of the polymorphisms, thus influencing the expected relationship between length and polymorphism. a major interest for evaluating the ssr markers in the carrot f populations was to assess their potential for mapping. linkage maps using some of these f2s have already been constructed and others are underway. these maps include different phenotypic traits of interest and -before this study- they were mainly constructed using anonymous dominant markers, such as aflps and rapds, with only very few markers, or none, in common, thus, making their comparative analyses and/or integration difficult. the present work identified ssrs that were polymorphic in two or more mapping populations, suggesting that these common markers may serve as anchoring points for merging carrot maps. besides the inclusion of ssr markers onto the carrot reference map, work is underway in our lab to include these polymorphic ssrs in other maps with different genetic backgrounds. the integration of carrot linkage maps would enhance their usefulness for assisting breeding of this species, by increasing marker saturation nearby genes of interest and thereby facilitating applications like positional gene cloning, among others. from our evaluation in seven carrot f families, ssr markers were polymorphic in at least one mapping population. because the pcr amplicons were size-separated using high-resolution agarose gel electrophoresis, which can only resolve fragments with size differences of at least bp, a fraction of the markers evaluated in some populations, generated ambiguous band patterns. although they may have been polymorphic, the bands were too close together in the gel to unambiguously score, and were classified as monomorphic. thus, if other fragment separation systems, with better resolution, are used, such as separation of fluorescently-labeled fragments through capillary electrophoresis, the number of polymorphic markers may be expanded in some populations, particularly in cases of dinucleotide ssr markers varying in a single repeat unit. high pcr amplification efficiencies were found in the f families for both sets of markers, gssrs and bssrs. comparable amplification efficiencies have been found in other plant species with ssr markers developed using hybridizations-based methods and sequence-based searches. transfer success of carrot ssrs across apiaceae the availability of ssr loci for economically important species has increased interest in primer transferability to related taxa, especially for species in which molecular resources are limited. in apiaceae, only a few publicly available ssrs have been reported previously, and these were developed from carrot and celery, the two most economically important crop species in the family. results from this study indicate that a significant fraction of carrot ssrs transfer successfully across apiaceae. locus amplification success was detected in to markers across non-carrot apiaceae species, including economically important crops like parsley, celery and cilantro. prospects of a broader utilization of these markers beyond carrot include their application in taxonomic, population, and conservation studies as well as for mapping and assisting breeding in crop species. it is, however, important to bear in mind that when using ssr markers across distantly-related species the amplification of a pcr product does not necessarily imply locus conservation, since size homoplasy, i.e. convergence in size of non-homologous fragments, may occur. considering the possibility of this source of confusion, verification of the pcr product identity by sequencing has been suggested previously, particularly when working across genera and if there is uncertainty regarding the size range of the amplicons obtained. however, verification through sequencing may not be necessary if working within the same genus as the species from which the ssrs markers were developed. thus, the use of carrot ssr markers for studies in non-daucus apiaceae should include verification, by sequencing, of the homology to the carrot ssr product sequence. transfer of carrot ssrs across daucus species was, in general, less successful than ssr transfer rate at the subgenus level reported for other species, whereas transfer of carrot ssrs across-genera was relatively higher than found in other plants. according to a previous review of ssr cross-transferability in plants, the average transferability across species in the same genus was %, and across related genera was %. we found these values to be % across daucus species and 41% across the apiaceae. however, it should be noted that ssr transfer success varied greatly across the different reports for both within-same genus and across different genera. the huge variation found across these studies likely reflects differences in phylogenetic distance between the source and target taxa within each family, as well as differences in the number of taxa and ssr loci analyzed, and in the type of sequences used for marker development. for example, est-derived ssrs are more conserved and thus they transfer across genera more readily than genomic ssrs ), among other factors. our data suggest generally a higher rate of success in amplifying carrot ssrs in plants more closely related to carrot. this should not be surprising since closer-related taxa have higher overall sequence homology which translates to more conserved ssr flanking regions and, therefore, easier transferability of primer pairs. negative relationships between ssr transfer success and phylogenetic distance between source and target taxa have been widely observed in many plant families. the potential usefulness of ssr markers for diversity and phylogenetic studies in apiaceae will depend, to a great extent, on the possibility that markers successfully amplify across different species and on the ability of the marker to detect polymorphism among the taxa. to have a preliminary picture of how suitable the ssr markers developed in this work may be for these applications, we investigated interspecific ssr variation among non-carrot species by analysis of amplicons sizes in the agarose gel images. thus, for each ssr, the total number of different alleles in the non-carrot species dataset was recorded. only ssrs that successfully amplified products in at least 80% of the non-carrot species were considered. overall, our results revealed ssrs that generated amplicons in most outside-carrot species. of these, ssrs produced 3- different alleles in the non-carrot group. it should be noted that our calculation of alleles/ssr in these selected markers is conservative, due to the low resolution of agarose gels which do not allow discrimination of different alleles varying in one or a few repeats. these results suggest that a significant proportion of the ssr markers developed herein may be suitable for addressing taxonomic or phylogenetic questions within apiaceae. further analysis of the ssrs that produced amplicons in the majority of the non-carrot taxa revealed interesting differences between the two ssr datasets. although more bssrs than gssrs amplified successfully in most non-carrot taxa, gssrs were much more polymorphic than bssrs at the interspecific level. for example, among gssrs markers produced or more different alleles, whereas only bssrs generated or more alleles/ssr. it is likely that the generally higher polymorphism of gssrs compared to bssrs at the inter-specific level, which is in agreement with our results for both sets of markers in the carrot f2s, may be also due to the higher number of repeat units present in gssrs. ssr linkage mapping prior to this work, important advances were made in the construction of carrot genetic maps with a range of molecular marker systems. although some rflps and a few scar and gene-specific markers were mapped, the most extensive genetic mapping data in carrot has been generated mainly with dominant aflp, rapd and transposon-display markers. while rflps are useful for comparative mapping purposes, high throughput genotyping and probe handling are difficult. similarly, the carotenoid genes mapped by just et al. are not as easily transferred to other mapping backgrounds since their analysis relied in most cases on snps, due to the lack of larger polymorphisms in these genes that can be scored as easily as ssrs. on the other hand, aflp, rapd and td markers, while providing a relatively large number of markers per assay and good genome coverage, have limited information content and are not of much use for comparative mapping purposes and for validating qtl across pedigrees. the addition of ssr markers to the carrot reference linkage map together with detailed characterization of this novel set of ssrs in subsets of six other mapping populations should allow significant advances in carrot comparative mapping and map-integration. the fact that most of the mapped ssrs were codominant in the b × qal-derived population, with 2- informative markers per linkage group, together with the identification of putative codominant ssrs in other mapping populations adds extra value to the data published here for pursuing these goals. the inclusion of ssrs in linkage maps with additional pedigrees is currently underway. the parental b map has a slightly larger total map length than the qal map. although the higher mean recombination found in b may help explain its larger map length, other factors -e.g., related to the type of markers used- may also cause this effect. different recombination frequencies can be obtained just by sampling of the different markers, as well as errors derived from calculations of genetic distances from dominant markers data. in the current map, we have modified linkage group designations and orientations, in accordance to recent cytogenetic data concerning the integration of carrot lgs with actual chromosomes. following standard conventions, consecutive numbers were assigned to the lgs in decreasing order of chromosome length, and four lgs were inverted in their north-south orientations to agree with the standard short arm/long arm presentation of their corresponding chromosomes. it must, however, be noted that although all the lgs could be unequivocally associated to chromosomes, and thus their number designations are correct and complete, unambiguous lg orientations could only be defined for six of the nine lgs. thus, chromosomes and in the current map, which correspond to former lgs and respectively, could not be unequivocally oriented, because a single anchored bac probe was used for lg-chromosome integrations. thus, their orientations were not modified from previous map versions. however, the possibility remains that future cytogenetic data may reveal different orientations for these lgs. these modifications based on recent cytogenetic data, and the addition of new ssr markers, add value to the updated reference carrot linkage map presented herein. overall, the current maps involve 193- mapped loci, including highly informative markers which consist of ssr, carotenoid gene and scar markers, spanning - cm, making it the most comprehensive genetic linkage map in the apiaceae to date. the ssr loci mapped across all lgs in both parental maps, and they were distributed fairly evenly within most individual lgs, thus recommending their usefulness as anchor points for merging carrot maps. in addition, such dispersed map distribution of the ssr loci, has allowed us to develop bac fish probes carrying ssr sequences mapped to specific lgs. these ssr-anchored probes were used for integrating some lgs of carrot with chromosomes by fish mapping. the positional association observed between ssrs and previously mapped genes suggests that these tandem repeats are frequent in genic regions of the genome. this is in agreement with results of morgante et al., demonstrating higher microsatellite frequencies in the transcribed and non-repetitive fractions of plant genomes. one ssr and two ssrs in lg and lg respectively, were located in the vicinity of two highly-significant quantitative trait loci for total root carotene accumulation. these correspond to the y and y loci, respectively, described by buishand and gabelman. microsatellites gssr and gssr although not tightly linked to y may be useful for marker assisted selection, either as a complement of, or as an alternative to the lack of amplification of other robust more-closely linked markers, such as y2mark.  this work reports on the development and characterization at various levels of a novel set of carrot ssr markers. analysis of the distribution of ssr motifs, repeat lengths and polymorphism across genomic and est sequences, as well as concerning different ssr isolation methods may help decide over future strategies for developing valuable ssr markers in this and other species. the genetic mapping of ssr loci onto the reference carrot linkage map, distributed throughout all linkage groups, together with the characterization of the entire set of markers in other mapping populations, should facilitate comparative mapping studies and integration of carrot maps. particularly important for these purposes are the codominant ssrs added to the map, which resulted in more than doubling the original number of informative markers in this -or any other- carrot map reported to date. in addition, ssr evaluations in carrot-related taxa indicates that a significant fraction of the carrot ssrs transfer successfully across apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. nonetheless, a fairly large number of potentially useful ssr markers were identified for non-carrot daucus and non-daucus apiaceae species, increasing the prospects of their successful utilization in other apiaceae. in addition, allelic diversity at selected ssr loci was evaluated using d. carota accessions. in this germplasm, the microsatellites proved to be highly polymorphic, with an average of alleles/locus and expected heterozygosity. the marker resources developed in this work should be a valuable tool for carrot breeding and genetics. 
this work reports on the development of carrot ssr markers and their characterization at various levels. thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for ssrs, and bioinformatic mining of ssrs in bac-end sequence and est sequence databases. the addition of ssrs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. the availability of informative, polymorphic, and robust pcr-based markers, such as microsatellites, will facilitate genetics and breeding of carrot and other apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. despite its importance, molecular resources in this species are relatively underdeveloped. ssr diversity evaluated in a collection of d. carota accessions revealed a high level of polymorphism for these selected loci, with an average of alleles/locus and expected heterozygosity. evaluation of microsatellites isolated from both dna sources in subsets of carrot f mapping populations revealed that ssrs from the hybridization-based method were longer, had more repeat units and were more polymorphic than ssrs isolated by sequence search. polymorphic markers in one family were evaluated in the entire f allowing the genetic mapping of ssrs onto the carrot reference map. in addition, ssr evaluations in carrot-related taxa indicated that a significant fraction of the carrot ssrs transfer successfully across apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. the apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~ million tons produced yearly worldwide. the ssr loci were distributed throughout all carrot linkage groups, with to ssrs/lg.
 the apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~ million tons produced yearly worldwide. despite its importance, molecular resources in this species are relatively underdeveloped. the availability of informative, polymorphic, and robust pcr-based markers, such as microsatellites, will facilitate genetics and breeding of carrot and other apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for ssrs, and bioinformatic mining of ssrs in bac-end sequence and est sequence databases. this work reports on the development of carrot ssr markers and their characterization at various levels.  evaluation of microsatellites isolated from both dna sources in subsets of carrot f mapping populations revealed that ssrs from the hybridization-based method were longer, had more repeat units and were more polymorphic than ssrs isolated by sequence search. overall, ssrs were polymorphic in at least one mapping population, and the percentage of polymophic ssrs across f populations ranged from to. polymorphic markers in one family were evaluated in the entire f allowing the genetic mapping of ssrs onto the carrot reference map. the ssr loci were distributed throughout all carrot linkage groups, with to ssrs/lg. in addition, ssr evaluations in carrot-related taxa indicated that a significant fraction of the carrot ssrs transfer successfully across apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. ssr diversity evaluated in a collection of d. carota accessions revealed a high level of polymorphism for these selected loci, with an average of alleles/locus and expected heterozygosity.  the addition of ssrs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. the markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other apiaceae. 