BACKGROUND
amorphophallus is a genus of perennial plants with an underground stem in the form of a corm and an umbrella-shaped leaf blade, which belongs to the family of araceae, order of alismatales  <cit> .
the genus includes more than  <dig> species, which are mainly distributed in the tropics or subtropics of west africa and south asia  <cit> .
amorphophallus has been cultivated and utilized in china as a medicinal herb and food source for 2000 years.
amorphophallus contains abundant water-soluble dietary fiber known as glucomannan, which is beneficial to the human health, possibly being responsible for lowering lipids, systolic blood pressure and glycemia  <cit> .
thus amorphophallus has great potential for commercial application.
the diploid species a. konjac <cit>  is widely planted in china, its corm contained high-level glucomannan and possessed wide adaptability, but poor disease resistance and low coefficient of propagation  <cit> ; in the contrast, a. bulbifer is a triploid   <cit>  with high disease resistance, characteristics of multi-seeding relay growth and high coefficient of propagation  <cit> .
however, molecular genetic research of amorphophallus is limited.
many studies have focused on extracting bioactive components  <cit> , and some studies have attempted to identify amorphophallus species taxonomically and to assess the genetic diversity of amorphophallus <cit> .
however, few genomic molecular makers have been developed until now: only  <dig> microsatellite  markers were exploited by santosa et al.
and pan et al.
<cit> .
ssr markers have been shown to be an effective tool to carry out germplasm characterization and genetic diversity studies  <cit> .
thus, more markers are needed for an in-depth understanding of the natural diversity of amorphophallus and to develop strategies for their sustainable utilization.
finally, as far as we know, gene discovery and molecular breeding of new amorphophallus economic cultivars have not been initiated because of the lack of genetic and genomic information.
ssr makers have been widely used in population genetics and crop breeding  <cit> .
according to the original sequences used for development of ssrs, ssrs were divided in two categories: genomic-ssrs, identified from random genomic sequences, and expressed sequence tag -ssrs, identified from transcribed rna sequences.
compared with est-ssrs, the development of genomic-ssrs is very expensive, labor intensive and timing consuming.
genomic-ssrs have uncertain linkage to the transcribed regions of the genome, while est-ssrs are potentially linked with particular transcriptional regions that contribute to agronomic phenotypes  <cit> .
in addition, est-ssrs have high transferability, because sequences containing est-ssrs are more conserved than sequences containing genomic-ssrs and can be utilized in other related species  <cit> .
large numbers of est sequences produced by deep sequencing technology have been deposited in the ncbi database and are being used for dna fingerprinting, comparative mapping, and evolutionary analysis  <cit> .
however, only a limited number of amorphophallus est sequence are available in the ncbi database.
here, we report the generation of a large expressed sequence dataset based on illumina hiseq™  <dig> sequencing data from the young leaves of two amorphophallus species, a. konjac and a. bulbifer.
in addition, we developed est-ssr markers and described their transferability in  <dig> species and  <dig> wild unknown “species”.
the relationship among cultivars and wild individuals was investigated using the developed est-ssrs.
RESULTS
assembly of amorphophallus transcriptome data from illumina sequencing
after stringent quality assessment and data filtering, illumina hiseq™  <dig> sequencing produced  <dig> , <dig> reads for a. konjac and  <dig> , <dig> reads for a. bulbifer with  <dig> % q <dig> bases and  <dig> % q <dig> bases , respectively .
the total length of the reads was about  <dig>  gigabases .
the raw data files are available in the sequence read archive at the national center for biotechnology information , with accession number sra <dig>  using the trinity assembler software  <cit> , short-read sequences from a. konjac and a. bulbifer were assembled into  <dig>  contigs and  <dig>  contigs, respectively .
the size distribution of these contigs is shown in figure  <dig>  little difference was found in the contig lengths from these two amorphophallus species; the contigs produced from both a. konjac and a. bulbifer were, on average, 276 bp long, with 381 bp and 372 bp median contig lengths , respectively.
with paired-end reads, contigs can be identified from the same transcript and the distance between these contigs can be estimated.
trinity can be used to map the reads back to the contigs, and to connect the contigs into unigenes that cannot be further extended either end.
as a result,  <dig>  and  <dig>  unigenes were obtained, with n <dig> values of  <dig> and 524 bp, from a. konjac and a. bulbifer, respectively .
to expand the utility of illumina sequencing data, the contigs from the two species were pooled and assembled into a non-redundant unigene set for further analysis.
as shown in table  <dig>   <dig>  non-redundant unigenes with an average length of 523 bp and an n <dig> of 635 bp, ranging from  <dig> to  <dig>  bp, were obtained.
the length distribution of two species and combined non-redundant unigenes are shown in figure  <dig>  the results revealed that the contig and unigene size distributions for two species were consistent, which implied the illumina sequencing solution was reliable and appropriate.
among combined non-redundant unigenes, the length of  <dig>   ranged from  <dig> to 500 bp,  <dig>  unigenes  ranged from  <dig> to 1000 bp, and  <dig>  unigenes  were more than 1000 bp in length.
frequency and distribution of different types of ssr markers
using a perl script known as misa,  <dig>  ssr loci were detected from  <dig>  non-redundant unigenes, representing  <dig> % of  <dig>  unigenes.
a total of  <dig>  unigene sequences contained more than one ssr loci.
in this study, the mononucleotide repeats were not considered.
the probability of ssrs from combined non-redundant unigenes was  <dig> % and the distribution density was one ssr loci per  <dig> kb.
dinucleotide repeats were the most common type, with a frequency of  <dig> % , followed by trinucleotide , tetranucleotide , pentanucleotide  and hexanucleotide repeats  .
the combined number of ssr loci from both dinucleotide and trinucleotide repeats was  <dig>  with a frequency of  <dig> %.
the frequencies of ssr distributed in different reiteration numbers are shown in figure 3b.
the number of ssr repeats ranged from  <dig> to  <dig>  and ssrs with six repeats were the most abundant, followed by those with five tandem repeats, seven tandem repeats and eight random repeats.
motifs that showed more than  <dig> reiterations were very rare, with a frequency of < <dig> %.
ssr loci with a length of 18 bp were the most frequent  followed by those with 15 bp , 12bp , 16bp  and 14 bp .
the longest ssr locus was 57 bp .
from  <dig>  ssr loci,  <dig> different repeat motifs, considering sequencing complementary, were identified.
the  <dig> most abundant ssr repeat motifs with different levels of repeats are shown in table  <dig>  the most abundant motifs were dinucleotide repeat units ag/ct, at/at and ac/gt with frequencies of  <dig> %,  <dig> % and  <dig> %, respectively.
among the trinucleotide repeat units, agg/cct, aag/ctt and ccg/cgg were the most abundant with frequencies of  <dig> %,  <dig> % and  <dig> %, respectively .
development, validation and transferability of ssr markers
based on combined non-redundant unigene sequences containing  <dig>  ssr loci,  <dig>  primer pairs  were designed using primer <dig>   <cit> .
primer pairs for the remaining  <dig>  loci could not be designed successfully because the sequences flanking the ssr loci were either too short or were not appropriate for designing primers.
from the  <dig>  primer pairs,  <dig> were randomly selected for primer synthesis and validation .
among the  <dig> primer pairs,  <dig> were successful in pcr amplification using genomic dna from a. konjac and a. bulbifer.
the remaining 50-pair primers failed to generate pcr products, even when the annealing temperature was reduced by 8°c.
of the  <dig> working primer pairs,  <dig> yielded pcr products of the expected fragment size.
the other  <dig> primer pairs generated multiple bands that did not include the target bands.
in addition,  <dig> primer pairs amplified fragments larger than the expected size, probably because of the presence of an insertion mutation.
only five primer pair amplified a shorter than expected fragment, suggesting that a deletion mutation had occurred in the amplified region.
to test the inter-species transferability, all  <dig> validated primer pairs, excluding the  <dig> pairs that generated multiple bands and nine pairs that generated more than 500 bp bands, were screened for polymorphism among  <dig> individual plants.
thirty five pairs of primers could amplify pcr products from all amorphophallus species .
twelve pair of primers only produced pcr fragments in a. bulbifer; seven pairs  amplified successful from a. bulbifer and a. bulbifer cultivars; four pairs  amplified successful from a. bulbifer, a. yuloensis and a. bulbifer cultivars; and twelve pairs amplified successful from a. konjac.
the remaining  <dig> primer pairs successfully amplified fragments from  <dig> to  <dig> species.
therefore, the est-ssrs developed from a. konjac and a. bulbifer could be successfully applied to other amorphophallus species, with a transferability rate of  <dig> %.
of  <dig> primers,  <dig> primers were monomorphic; the other  <dig> primers were polymorphic; the proportion of polymorphic primers was  <dig> %.
from the  <dig> polymorphic loci, the number of alleles per locus ranged from  <dig> to  <dig> alleles.
a total of  <dig>  alleles were identified, with an average of  <dig>  alleles per locus.
across  <dig> loci, the polymorphic information content  ranged from  <dig>  for ak-est-ssr <dig> to  <dig>  for ak-est-ssr <dig> , suggesting that the developed est-ssrs were highly polymorphic.
to determine the likely function of these est-ssrs, they were subjected to blast analysis.
the results showed that  <dig> transcriptome sequences from the  <dig> polymorphic ssr makers shared significant homology to other plants functional loci.
the functional annotations of the developed markers are listed in additional file  <dig>
the genetic diversity and cluster analysis in genus amorphophallus
the  <dig> pair primers that yielded clear, highly polymorphic bands were used to assess the genetic diversity in a set of  <dig> individual plants representing diverse wild unknown species and cultivated genotypes of amorphophallus .
a phenogram tree based on jaccard’s similarity coefficients was constructed, which showed three distinct clusters at a cut-off similarity index of  <dig>  .
cluster i contained five species and five unknown “species”, and was divided into three sub-clusters: ia, ib and ic, at a cut-off similarity index of  <dig> .
sub-cluster ia comprised two wild a. konjac, three a. konjac cultivar genotypes, three yunnan species and three unknown “species”.
sub-cluster ib comprised two unknown “species” from chongqing and yunan, respectively.
sub-cluster ic comprised one wild a. konjac and one a. kiusianus.
the wild a. konjac species from kunming was distant from the other five a. konjac species.
cluster ii included seven species and it was divided into three sub-clusters iia, iib and iic, at a cut-off similarity index of  <dig> .
sub-cluster iia comprised one a. bulbifer and one a. bulbifer cultivar; sub-cluster iib comprised one hunan species and two yunnan species; sub-cluster iic comprised three species from yunnan.
cluster iii contained a. albus and a. paeoniifolius from yunan and chongqing, respectively.
* a. sp means a wild unknown “species”  with no clear classification.
discussion
ssr marker frequency and distribution in amorphophallus transcriptome
genetic markers are important for studying population genetic structure, diversity analysis and the genetic basis of adaptive traits  <cit> .
deep transcriptome sequencing provides a good resource for the development of ssrs because of the enormous amount of sequence of data that it generates.
markers based on transcriptome sequences are useful for the detection of functional variation and gene associated genetic analysis  <cit> .
based on the  <dig>  non-redundant unigene sequences, a total of  <dig>  ssrs were identified.
about  <dig> % of the transcriptomic sequences possess ssr loci.
this rate is higher than wheat , barley  and epimedium , but lower than coffee   <cit> .
the est-ssrs frequency is dependent on the search parameters for exploring ssr markers, e.g.
the repeat length threshold and the number of repeat motifs unit.
in our study, the mononucleotide repeat motifs were excluded for identifying ssrs because they can result from sequencing errors.
in addition, the software used to detect ssr loci can affect the ssr frequency.
some tools can detect imperfect ssrs, e.g.
sputnik, while ssrit and misa  can only detect perfect ssrs.
the distribution density in amorphophallus is one ssr loci per  <dig>  kb, compared with  <dig>  kb in rice,  <dig>  kb in wheat,  <dig>  kb in soybean, 14 kb in arabidopsis, and 20 kb in cotton  <cit> .
the difference in ssr density could partially depend on the minimum length of the ssr repeat motif  <cit> .
in addition, varshney et al.
assumed that the high frequency of ssr in rice est sequences might be due to its small genome size  <cit> .
dinucleotide repeat motifs are the most frequent repeat type among the amorphophallus unigenes analyzed, representing  <dig> % of the ssr loci identified, followed by trinucleotide , tetranucleotide , pentanucleotide  and hexanucleotide  .
this is consistent with the est-ssrs distributions reported in pigeonpea, peach, spruce, pumpkin, kiwifruit and coffee, where the dinucleotide motif is the most frequent  <cit> .
as shown in table  <dig>  the most abundant dinucleotide repeat motif was ag/ct  in our transcriptome sequences.
the same results were also found in other plant species  <cit> .
the most abundant trinucleotide repeat motif was agg/cct , closely followed by aag/ctt .
these results agree with the report from poncet et al.
<cit> .
unlike the results obtained in amorphophallus, the most frequency trinucleotide repeat motifs were aac/ttg in wheat, aag/ctt in soybean and epimedium sagittatum, ccg/ggc in maize, barley and sorghum  <cit> .
the previous studies suggested that the trinucleotide aag/ctt is a common motif and that ccg/cgg is very rare in dicotyledonous plants.
interestingly, the trinucleotide ccg/cgg motif is common in monocots  <cit> .
in this study, the trinucleotide ccg/cgg motif  is the third most common repeat type, closely followed by agg/cct  and aag/ctt .
these results strongly support the view that the abundance ccg/cgg motif is a specific feature of monocot genomes, which could have resulted from the high gc content and consequent codon usage bias  <cit> .
polymorphism and genera transferability of ssr markers
est-ssrs are very useful and attractive because they are located in coding regions of the genome.
therefore, they often show a high degree of transferability to related species  <cit> .
in this study,  <dig> designed primer pairs were used for validation of the est-ssr markers in amorphophallus,  <dig> primer pairs  yielded amplicons in a. konjac or a. bulbifer.
this result was similar to the success rate of 60-90% amplification in previous studies  <cit> .
from  <dig> primer pairs, we obtained  <dig> polymorphic est-ssr markers within  <dig> samples, with the polymorphic proportion of  <dig> %.
compared with other plants, the polymorphic ratio of est-ssrs in amorphophallus is very high  <cit> .
using these polymorphic markers,  <dig>  alleles were identified, with an average of  <dig>  alleles per locus in  <dig> individual plants.
pic value ranges from  <dig>  to  <dig> .
santosa et al.
reported  <dig> polymorphic ssr markers with an average of  <dig>  alleles per locus in a. paeoniifolius <cit> .
santosa et al.
also reported ten ssr markers with an average of  <dig>  gene diversity  in  <dig> a. paeoniifolius individual plants  <cit> .
the transferability of est-ssrs among different related genera has been reported in many crop plants.
ellis et al.
summarized the transferablility of est-ssrs and showed that the ratio of est-ssrs cross-genera transferability ranged from 10% to 90%  <cit> .
in this study, the amorphophallus species had a high transferability rate .
this high success rate indicated that the different species in the genus amorphophallus may evolutionarily closely related.
evaluation of genetic relationships among different species of amorphophallus
collection of germplasm has been carried out in china, india and japan.
however, until now, evaluation of the genetic diversity of amorphophallus germplasm has been limited.
in this study, three major groups were identified at a cut-off similarity index of  <dig> .
the level of genetic similarity  was revealed in the  <dig> individual plants, which included  <dig> species and  <dig> wild unknown “species”, using  <dig> polymorphic primers.
the genetic variation between a. konjac and a. paeoniifolius was the highest among species in amorphophallus.
the intra-specific variation in a. konjac species is higher than that of some inter-species, and the a. konjac wild genotypes were clustered with cultivated genotypes.
these results implied that amorphophallus has a rich genetic diversity; the variation between wild and cultivar genotypes in a. konjac species was very small.
this may be because the cultivars have been directly derived from the wild species without systematic genetic breeding.
in addition, five unknown amorphophallus “species” were used to evaluate their taxonomic status.
21-a.
sp from chongqing, 24-a.
sp from hubei and 25-a.
sp from myanmar were clustered in one group with a. krauseiengler and a. tokinensis.
20-a.sp and 12-a.sp comprised one group: sub-cluster ib.
evaluation of the genetic status of these unknown species is valuable for utilizing new resources and for breeding.
sedayu et al.
reported that the molecular phylogeny of the genus amorphophallus contained  <dig> taxa, based on a combined analysis of trnl, rbcl and leafy second intron sequences, which reflected the biogeographical distribution of amorphophallus <cit> .
grob et al.
also reported the phylogeny of  <dig> amorphophallus using the nuclear floricaula/leafy second intron  <cit> .
our results complement and support these previous reports.
CONCLUSIONS
a large-scale transcriptome dataset with  <dig>  unigenes from a. konjac and a. bulbifer is reported.
a total of  <dig>  primer pairs were successfully designed based on the transcriptome sequences.
three hundred and twenty primer pairs from the combined non-redundant unigenes of the two sequenced amorphophallus species were validated.
using the validated primers, the diversity of  <dig> individual plants containing  <dig> species and  <dig> wild unknown “species” was analyzed.
the developed ssr markers are a valuable resource for studying genetic diversity, linkage mapping, and germplasm characterization analysis in amorphophallus .
the esr-ssr markers were developed based on conserved expressed sequences; therefore, they may be applied to other species because of their high transferability.
to the best of our knowledge, this is the first attempt to exploit a transcriptome database and develop a large set of ssr markers in amorphophallus.

 amorphophallus is a genus of perennial plants with an underground stem in the form of a corm and an umbrella-shaped leaf blade, which belongs to the family of araceae, order of alismatales. the genus includes more than species, which are mainly distributed in the tropics or subtropics of west africa and south asia. amorphophallus has been cultivated and utilized in china as a medicinal herb and food source for 2000 years. amorphophallus contains abundant water-soluble dietary fiber known as glucomannan, which is beneficial to the human health, possibly being responsible for lowering lipids, systolic blood pressure and glycemia. thus amorphophallus has great potential for commercial application. the diploid species a. konjac is widely planted in china, its corm contained high-level glucomannan and possessed wide adaptability, but poor disease resistance and low coefficient of propagation; in the contrast, a. bulbifer is a triploid with high disease resistance, characteristics of multi-seeding relay growth and high coefficient of propagation. however, molecular genetic research of amorphophallus is limited. many studies have focused on extracting bioactive components, and some studies have attempted to identify amorphophallus species taxonomically and to assess the genetic diversity of amorphophallus. however, few genomic molecular makers have been developed until now: only microsatellite markers were exploited by santosa et al. and pan et al.. ssr markers have been shown to be an effective tool to carry out germplasm characterization and genetic diversity studies. thus, more markers are needed for an in-depth understanding of the natural diversity of amorphophallus and to develop strategies for their sustainable utilization. finally, as far as we know, gene discovery and molecular breeding of new amorphophallus economic cultivars have not been initiated because of the lack of genetic and genomic information. ssr makers have been widely used in population genetics and crop breeding. according to the original sequences used for development of ssrs, ssrs were divided in two categories: genomic-ssrs, identified from random genomic sequences, and expressed sequence tag -ssrs, identified from transcribed rna sequences. compared with est-ssrs, the development of genomic-ssrs is very expensive, labor intensive and timing consuming. genomic-ssrs have uncertain linkage to the transcribed regions of the genome, while est-ssrs are potentially linked with particular transcriptional regions that contribute to agronomic phenotypes. in addition, est-ssrs have high transferability, because sequences containing est-ssrs are more conserved than sequences containing genomic-ssrs and can be utilized in other related species. large numbers of est sequences produced by deep sequencing technology have been deposited in the ncbi database and are being used for dna fingerprinting, comparative mapping, and evolutionary analysis. however, only a limited number of amorphophallus est sequence are available in the ncbi database. here, we report the generation of a large expressed sequence dataset based on illumina hiseq™ sequencing data from the young leaves of two amorphophallus species, a. konjac and a. bulbifer. in addition, we developed est-ssr markers and described their transferability in species and wild unknown “species”. the relationship among cultivars and wild individuals was investigated using the developed est-ssrs.  assembly of amorphophallus transcriptome data from illumina sequencing after stringent quality assessment and data filtering, illumina hiseq™ sequencing produced, reads for a. konjac and, reads for a. bulbifer with % q bases and % q bases, respectively. the total length of the reads was about gigabases. the raw data files are available in the sequence read archive at the national center for biotechnology information, with accession number sra using the trinity assembler software, short-read sequences from a. konjac and a. bulbifer were assembled into contigs and contigs, respectively. the size distribution of these contigs is shown in figure  little difference was found in the contig lengths from these two amorphophallus species; the contigs produced from both a. konjac and a. bulbifer were, on average, 276 bp long, with 381 bp and 372 bp median contig lengths, respectively. with paired-end reads, contigs can be identified from the same transcript and the distance between these contigs can be estimated. trinity can be used to map the reads back to the contigs, and to connect the contigs into unigenes that cannot be further extended either end. as a result, and unigenes were obtained, with n values of and 524 bp, from a. konjac and a. bulbifer, respectively. to expand the utility of illumina sequencing data, the contigs from the two species were pooled and assembled into a non-redundant unigene set for further analysis. as shown in table  non-redundant unigenes with an average length of 523 bp and an n of 635 bp, ranging from to  bp, were obtained. the length distribution of two species and combined non-redundant unigenes are shown in figure  the results revealed that the contig and unigene size distributions for two species were consistent, which implied the illumina sequencing solution was reliable and appropriate. among combined non-redundant unigenes, the length of ranged from to 500 bp, unigenes ranged from to 1000 bp, and unigenes were more than 1000 bp in length. frequency and distribution of different types of ssr markers using a perl script known as misa, ssr loci were detected from non-redundant unigenes, representing % of unigenes. a total of unigene sequences contained more than one ssr loci. in this study, the mononucleotide repeats were not considered. the probability of ssrs from combined non-redundant unigenes was % and the distribution density was one ssr loci per kb. dinucleotide repeats were the most common type, with a frequency of %, followed by trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeats. the combined number of ssr loci from both dinucleotide and trinucleotide repeats was with a frequency of %. the frequencies of ssr distributed in different reiteration numbers are shown in figure 3b. the number of ssr repeats ranged from to and ssrs with six repeats were the most abundant, followed by those with five tandem repeats, seven tandem repeats and eight random repeats. motifs that showed more than reiterations were very rare, with a frequency of < %. ssr loci with a length of 18 bp were the most frequent followed by those with 15 bp, 12bp, 16bp and 14 bp. the longest ssr locus was 57 bp. from ssr loci, different repeat motifs, considering sequencing complementary, were identified. the most abundant ssr repeat motifs with different levels of repeats are shown in table  the most abundant motifs were dinucleotide repeat units ag/ct, at/at and ac/gt with frequencies of %, % and %, respectively. among the trinucleotide repeat units, agg/cct, aag/ctt and ccg/cgg were the most abundant with frequencies of %, % and %, respectively. development, validation and transferability of ssr markers based on combined non-redundant unigene sequences containing ssr loci, primer pairs were designed using primer. primer pairs for the remaining loci could not be designed successfully because the sequences flanking the ssr loci were either too short or were not appropriate for designing primers. from the primer pairs, were randomly selected for primer synthesis and validation. among the primer pairs, were successful in pcr amplification using genomic dna from a. konjac and a. bulbifer. the remaining 50-pair primers failed to generate pcr products, even when the annealing temperature was reduced by 8°c. of the working primer pairs, yielded pcr products of the expected fragment size. the other primer pairs generated multiple bands that did not include the target bands. in addition, primer pairs amplified fragments larger than the expected size, probably because of the presence of an insertion mutation. only five primer pair amplified a shorter than expected fragment, suggesting that a deletion mutation had occurred in the amplified region. to test the inter-species transferability, all validated primer pairs, excluding the pairs that generated multiple bands and nine pairs that generated more than 500 bp bands, were screened for polymorphism among individual plants. thirty five pairs of primers could amplify pcr products from all amorphophallus species. twelve pair of primers only produced pcr fragments in a. bulbifer; seven pairs amplified successful from a. bulbifer and a. bulbifer cultivars; four pairs amplified successful from a. bulbifer, a. yuloensis and a. bulbifer cultivars; and twelve pairs amplified successful from a. konjac. the remaining primer pairs successfully amplified fragments from to species. therefore, the est-ssrs developed from a. konjac and a. bulbifer could be successfully applied to other amorphophallus species, with a transferability rate of %. of primers, primers were monomorphic; the other primers were polymorphic; the proportion of polymorphic primers was %. from the polymorphic loci, the number of alleles per locus ranged from to alleles. a total of alleles were identified, with an average of alleles per locus. across loci, the polymorphic information content ranged from for ak-est-ssr to for ak-est-ssr, suggesting that the developed est-ssrs were highly polymorphic. to determine the likely function of these est-ssrs, they were subjected to blast analysis. the results showed that transcriptome sequences from the polymorphic ssr makers shared significant homology to other plants functional loci. the functional annotations of the developed markers are listed in additional file the genetic diversity and cluster analysis in genus amorphophallus the pair primers that yielded clear, highly polymorphic bands were used to assess the genetic diversity in a set of individual plants representing diverse wild unknown species and cultivated genotypes of amorphophallus. a phenogram tree based on jaccard’s similarity coefficients was constructed, which showed three distinct clusters at a cut-off similarity index of. cluster i contained five species and five unknown “species”, and was divided into three sub-clusters: ia, ib and ic, at a cut-off similarity index of. sub-cluster ia comprised two wild a. konjac, three a. konjac cultivar genotypes, three yunnan species and three unknown “species”. sub-cluster ib comprised two unknown “species” from chongqing and yunan, respectively. sub-cluster ic comprised one wild a. konjac and one a. kiusianus. the wild a. konjac species from kunming was distant from the other five a. konjac species. cluster ii included seven species and it was divided into three sub-clusters iia, iib and iic, at a cut-off similarity index of. sub-cluster iia comprised one a. bulbifer and one a. bulbifer cultivar; sub-cluster iib comprised one hunan species and two yunnan species; sub-cluster iic comprised three species from yunnan. cluster iii contained a. albus and a. paeoniifolius from yunan and chongqing, respectively. * a. sp means a wild unknown “species” with no clear classification. discussion ssr marker frequency and distribution in amorphophallus transcriptome genetic markers are important for studying population genetic structure, diversity analysis and the genetic basis of adaptive traits. deep transcriptome sequencing provides a good resource for the development of ssrs because of the enormous amount of sequence of data that it generates. markers based on transcriptome sequences are useful for the detection of functional variation and gene associated genetic analysis. based on the non-redundant unigene sequences, a total of ssrs were identified. about % of the transcriptomic sequences possess ssr loci. this rate is higher than wheat, barley and epimedium, but lower than coffee. the est-ssrs frequency is dependent on the search parameters for exploring ssr markers, e.g. the repeat length threshold and the number of repeat motifs unit. in our study, the mononucleotide repeat motifs were excluded for identifying ssrs because they can result from sequencing errors. in addition, the software used to detect ssr loci can affect the ssr frequency. some tools can detect imperfect ssrs, e.g. sputnik, while ssrit and misa can only detect perfect ssrs. the distribution density in amorphophallus is one ssr loci per  kb, compared with  kb in rice,  kb in wheat,  kb in soybean, 14 kb in arabidopsis, and 20 kb in cotton. the difference in ssr density could partially depend on the minimum length of the ssr repeat motif. in addition, varshney et al. assumed that the high frequency of ssr in rice est sequences might be due to its small genome size. dinucleotide repeat motifs are the most frequent repeat type among the amorphophallus unigenes analyzed, representing % of the ssr loci identified, followed by trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide. this is consistent with the est-ssrs distributions reported in pigeonpea, peach, spruce, pumpkin, kiwifruit and coffee, where the dinucleotide motif is the most frequent. as shown in table  the most abundant dinucleotide repeat motif was ag/ct in our transcriptome sequences. the same results were also found in other plant species. the most abundant trinucleotide repeat motif was agg/cct, closely followed by aag/ctt. these results agree with the report from poncet et al.. unlike the results obtained in amorphophallus, the most frequency trinucleotide repeat motifs were aac/ttg in wheat, aag/ctt in soybean and epimedium sagittatum, ccg/ggc in maize, barley and sorghum. the previous studies suggested that the trinucleotide aag/ctt is a common motif and that ccg/cgg is very rare in dicotyledonous plants. interestingly, the trinucleotide ccg/cgg motif is common in monocots. in this study, the trinucleotide ccg/cgg motif is the third most common repeat type, closely followed by agg/cct and aag/ctt. these results strongly support the view that the abundance ccg/cgg motif is a specific feature of monocot genomes, which could have resulted from the high gc content and consequent codon usage bias. polymorphism and genera transferability of ssr markers est-ssrs are very useful and attractive because they are located in coding regions of the genome. therefore, they often show a high degree of transferability to related species. in this study, designed primer pairs were used for validation of the est-ssr markers in amorphophallus, primer pairs yielded amplicons in a. konjac or a. bulbifer. this result was similar to the success rate of 60-90% amplification in previous studies. from primer pairs, we obtained polymorphic est-ssr markers within samples, with the polymorphic proportion of %. compared with other plants, the polymorphic ratio of est-ssrs in amorphophallus is very high. using these polymorphic markers, alleles were identified, with an average of alleles per locus in individual plants. pic value ranges from to. santosa et al. reported polymorphic ssr markers with an average of alleles per locus in a. paeoniifolius. santosa et al. also reported ten ssr markers with an average of gene diversity in a. paeoniifolius individual plants. the transferability of est-ssrs among different related genera has been reported in many crop plants. ellis et al. summarized the transferablility of est-ssrs and showed that the ratio of est-ssrs cross-genera transferability ranged from 10% to 90%. in this study, the amorphophallus species had a high transferability rate. this high success rate indicated that the different species in the genus amorphophallus may evolutionarily closely related. evaluation of genetic relationships among different species of amorphophallus collection of germplasm has been carried out in china, india and japan. however, until now, evaluation of the genetic diversity of amorphophallus germplasm has been limited. in this study, three major groups were identified at a cut-off similarity index of. the level of genetic similarity was revealed in the individual plants, which included species and wild unknown “species”, using polymorphic primers. the genetic variation between a. konjac and a. paeoniifolius was the highest among species in amorphophallus. the intra-specific variation in a. konjac species is higher than that of some inter-species, and the a. konjac wild genotypes were clustered with cultivated genotypes. these results implied that amorphophallus has a rich genetic diversity; the variation between wild and cultivar genotypes in a. konjac species was very small. this may be because the cultivars have been directly derived from the wild species without systematic genetic breeding. in addition, five unknown amorphophallus “species” were used to evaluate their taxonomic status. 21-a. sp from chongqing, 24-a. sp from hubei and 25-a. sp from myanmar were clustered in one group with a. krauseiengler and a. tokinensis. 20-a.sp and 12-a.sp comprised one group: sub-cluster ib. evaluation of the genetic status of these unknown species is valuable for utilizing new resources and for breeding. sedayu et al. reported that the molecular phylogeny of the genus amorphophallus contained taxa, based on a combined analysis of trnl, rbcl and leafy second intron sequences, which reflected the biogeographical distribution of amorphophallus. grob et al. also reported the phylogeny of amorphophallus using the nuclear floricaula/leafy second intron. our results complement and support these previous reports.  a large-scale transcriptome dataset with unigenes from a. konjac and a. bulbifer is reported. a total of primer pairs were successfully designed based on the transcriptome sequences. three hundred and twenty primer pairs from the combined non-redundant unigenes of the two sequenced amorphophallus species were validated. using the validated primers, the diversity of individual plants containing species and wild unknown “species” was analyzed. the developed ssr markers are a valuable resource for studying genetic diversity, linkage mapping, and germplasm characterization analysis in amorphophallus. the esr-ssr markers were developed based on conserved expressed sequences; therefore, they may be applied to other species because of their high transferability. to the best of our knowledge, this is the first attempt to exploit a transcriptome database and develop a large set of ssr markers in amorphophallus. 
amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of west africa and south asia. a large number of molecular markers are required for genetic diversity study and improving disease resistance in amorphophallus. genetic diversity analysis was done using highly polymorphic ssr markers. the large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in amorphophallus. its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. cdnas of a. konjac and a. bulbifer were sequenced using illumina hiseq™ sequencing technology. here, we report large scale of transcriptome sequencing of two species: amorphophallus konjac and amorphophallus bulbifer using deep sequencing technology, and microsatellite markers were identified based on these transcriptome sequences. of these, primers were synthesized and used for validation of amplification and assessment of polymorphisms in individual plants.
 amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of west africa and south asia. its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. genetic studies of amorphophallus have been hindered by a lack of genetic markers. a large number of molecular markers are required for genetic diversity study and improving disease resistance in amorphophallus. here, we report large scale of transcriptome sequencing of two species: amorphophallus konjac and amorphophallus bulbifer using deep sequencing technology, and microsatellite markers were identified based on these transcriptome sequences.  cdnas of a. konjac and a. bulbifer were sequenced using illumina hiseq™ sequencing technology. a total of non-redundant unigenes were assembled from about gigabases, and ssrs were identified in non-redundant unigenes. di-nucleotide ssrs were the most abundant motif, followed by tri-, tetra-, penta-, and hexa-nucleotides repeats. the top di- and tri-nucleotide repeat motifs included ag/ct and agg/cct, respectively. a total of primer pairs were designed for marker development. of these, primers were synthesized and used for validation of amplification and assessment of polymorphisms in individual plants. the total of primer pairs yielded pcr amplification products, of which were polymorphic. the number of alleles ranged from to and the polymorphism information content valued ranged from to. genetic diversity analysis was done using highly polymorphic ssr markers. a phenogram based on jaccard’s similarity coefficients was constructed, which showed a distinct cluster of amorphophallus individuals.  a total of ssr markers have been identified in amorphophallus using transcriptome sequencing. one hundred and seventy-seven polymorphic markers were successfully validated in individuals. the large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in amorphophallus. amorphophallusmicrosatellite markertranscriptomegenetic diversity 