pekinensis ) is a diploid ( 2n = 2x = 20 ) dicot with a genomic size of 550  mb ( http://www.brassica.info/resource/ ) .
the species originated in china and now has become one of the most important and widely cultivated leaf vegetables in asia .
the tight leafy head is the main edible part . after a long history of domestication
, chinese cabbage evolves into different cultivars with a variety of characteristics , such as rosette leaf morphology , heading leaf morphology , leafy head shape , size , and structure , flowering time , nutrient composition , and resistance to biotic and abiotic .
a better understanding of the molecular mechanism of evolution of chinese cabbage and further development of marker - assisted selection ( mas ) will accelerate the selection process of improved cultivars to meet the growing consumers and environmental needs .
although progress has been made in underlining the molecular mechanism [ 25 ] , many aspects are still unclear .
molecular markers have been widely used to study the genetic basis of important traits and map regulatory genes in plants .
markers tightly linked with important agronomic traits can potentially be used for molecular breeding to develop improved cultivars .
many molecular markers and genetic maps of chinese cabbage have been reported previously [ 625 ] .
however , there is still a great need to develop novel molecular markers for construction of high - density linkage maps for genetics and molecular studies of important traits in chinese cabbage .
simple sequence repeat ( ssr ) markers or microsatellite markers are among the most important markers in plants .
ssrs have been widely used in genetic mapping and molecular breeding in plants because they are highly abundant and have significant polymorphism .
other factors , like accessibility for detection , reliability , and codominance , also make them perfect markers for such purposes .
ssrs found in transcribed sequences are called expressed sequence - simple sequence repeats ( est - ssrs ) . compared with genomic - ssrs detected in noncoding sequences ,
est - ssrs are more efficient for qtl mapping , gene targeting , and mas .
as transcribed sequences are more conserved than noncoding sequences , the transferability of est - ssrs is better than genomic - ssrs [ 2830 ] , which can be utilized for cross genome comparison and evolutionary analysis [ 27 , 31 ] .
additionally , abundant ests were generated in recent years with the development of next - generation sequencing approaches , making identification of est - ssrs more practical and cost - efficient .
many est - ssrs have been identified in chinese cabbage [ 16 , 20 , 25 , 3336 ] . because whole genome sequencing of chinese cabbage is still underway , new est - ssrs
could also be identified for further studies such as high - density genetic linkage map construction , gene / qtl mapping , and cultivar identification . in our previous study , the whole transcriptomes were analyzed for the rosette leaves and folding leaves of a typical heading chinese cabbage , namely , fushanbaotou , using a solexa / illumina rna - seq platform , and a large - scale est database was generated . in this study , we further assembled those ests from the rl and fl libraries into nonredundant unigenes .
a total of 10,420 est - ssrs were identified , among which 2744 est - ssrs are detected for the first time , according to the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .
we characterized these identified est - ssrs and designed 7877 pcr primer pairs for 1561 est - ssrs .
furthermore , serving as a validation purpose , we tested polymorphisms of 24 est - ssrs .
we expect this study can pave the road for further investigation of new est - ssr markers and for construction of high - density genetic maps .
for est - ssr identification and primer design , a typical heading chinese cabbage , namely , fushanbaotou , was used in this study . for primer assessment and ssr polymorphism analysis ,
a panel of twenty - four cultivars of chinese cabbage was used , including nineteen morphologically diverse cultivars of brassica rapa l. ssp .
pekinensis ( b. pekinensis l. ) and five brassica rapa l. chinensis ( b. chinensis l. ) .
leaves were collected after they were grown for two weeks from ten seedlings of each cultivar and were pooled together for dna extraction .
we assembled the clean read dataset presented by wang et al .   from the rl and fl libraries according to the methods described by wang et al .   using the trinity software ( http://trinityrnaseq.sourceforge.net/ ) .
redundant sequences were removed and overlapping unigenes were assembled into continuous sequences by the tigr gene indices clustering ( tgicl ) tools .
similarity was set at 94% and an overlap length was set at 100  bp .
parameters were set with a minimum number of 12 , 6 , 5 , 5 , 4 , and 4 repeat units for identification of mono- , di- , tri- , tetra- , penta- , and hexanucleotide motifs , respectively .
primer length ranged from 18 to 28  bp ( with an optimality at 23 ) .
the physical positions of the est - ssrs identified in the study were determined by aligning the ssrs and flanking sequences ( 50  bp at each side ) to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn .
new est - ssrs were identified by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .
the dna sample of the chinese cabbage fushanbaotou was used as template to detect the availability of ssr primers designed above .
the dna samples of those aforementioned twenty - four cultivars of chinese cabbage were used as templates for ssr polymorphism analysis .
the polymorphisms of est - ssrs were validated by 6% denaturing polyacrylamide gel electrophoresis , 12% nondenaturing polyacrylamide gel electrophoresis , and sequencing .
high quality clean read data from the rl and fl libraries by wang et al .  
a total of 99,684 and 95,411 contigs were obtained , with an average length of 333 and 342  bp and a median length ( n50 ) of 531 and 536  bp , from the rl and fl libraries , respectively ( table 1 ) .
contigs from the same transcript were detected with paired - end reads , as well as the distances between these contigs . using the trinity software package , we assembled these contigs into unigenes , in which ns were removed .
a total of 46,294 and 48,473 unigenes from the rl and fl libraries were obtained with an average length of 707 and 680  bp and a median length ( n50 ) of 1000 and 980  bp , respectively ( table 1 ) .
size distribution of the contigs and unigenes is consistent with the rl and fl libraries as shown in figure 1 , indicating that our illumina sequencing solution is reliable and reproducible .
unigenes from the two samples were combined ; redundant unigenes were removed ; and the rest was assembled with tgicl   to form a single dataset , which represents 40.7  mb of sequence and contains a total of 51,694 nonredundant unigenes , with an average read length of 788  bp , and a median read length ( n50 ) of 1154  bp ( table 1 ) .
the sequences of the unigenes are listed in table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/473028 ) .
the length of 24,271 nonredundant unigenes ( 46.95% ) is between 200 and 500  bp ; the length of 13,613 ( 26.33% ) is between 501 and 1,000  bp , and the length of 13,810 ( 26.72% ) is longer than 1,000  bp ( figure 1 ) .
a total of 10420 est - ssrs were detected with the microsatellite software ( misa ; http://pgrc.ipk-gatersleben.de/misa/ ) in 8571 unigenes , accounting for 16.6% of total nonredundant unigenes ( tables 2 and s2 ) .
the mean ssr density is one per 3.9  kb , corresponding to one for every 5.0 nonredundant unigenes .
1502 unigenes ( 17.5% ) harbored more than one ssr and 666 ssrs ( 6.4% ) were present in compound formation that had more than one repeat type ( table 2 ) .
the ssrs with tri- and dinucleotide repeat motifs were the most common ( 4,405 , 42.27% ; 4,043 , 38.80% , resp . ) , followed by mono- ( 1,644 , 15.78% ) , hexa- ( 126 , 1.21% ) , penta- ( 112 , 1.07% ) and tetra- ( 90 , 0.86% ) nucleotide repeat motifs ( figure 2 ) .
the most common two repeat motif types accounted for 81.07% of the total ssrs detected , and the rest repeat motifs types only accounted for 18.93% .
the iterate number of repeat units in an est - ssr ranged from 4 to 25 .
the occurrence frequency of est - ssts with different iterate numbers was found to be unequal either .
est - ssrs with iterate number of 5 ( 2832 , 27.18% ) were the most common ones , followed by 6 ( 2739 , 26.29% ) , 7 ( 1368 , 13.13% ) , 8 ( 703 , 6.75% ) , 12 ( 542 , 5.20% ) , and 9 ( 480 , 4.61% ) ( table s3 ) .
a dinucleotide containing est - ssrs with a maximum of 25 repeat units was identified . for est - ssrs with more than 10 repeat units ,
the mononucleotide repeat motifs were the most abundant , accounting for 93.46% of these est - ssrs .
the lengths of est - ssr sequences ranged from 12 to 65  bp ( table s4 ) .
the longest one is a pentanucleotide containing est - ssr with 65  bp in length .
the lengths of most est - ssrs are from 12 to 20  bp , accounting for 91.47% of the total est - ssrs , followed by est - ssrs with 2130  bp in length ( 874 ssrs , 8.39% ) .
only 13 est - ssrs were identified with over 30  bp , accounting for 0.12% of the total est - ssrs .
a total of 124 est - ssr motifs were identified , including 2 mono- , 3 di- , 10 tri- , 13 tetra- , 33 penta- , and 63 hexanucleotide repeat units containing est - ssrs .
the dominant motif identified in our est - ssrs was ag / ct ( 3,519 , 33.8% ) , followed by a / t ( 1,562 , 15.0% ) , aag / ctt ( 1,445 , 13.9% ) , agg / cct ( 776 , 7.4% ) , atc / atg ( 627 , 6.0% ) , aac / gtt ( 392 , 4.4% ) , acc / ggt ( 392 , 3.8% ) , ac / gt ( 349 , 3.3% ) , and agc / ctg ( 317 , 3.0% ) ( figure 3 ) .
the other 115 motifs have low frequency , accounting only for 9.3% of total est - ssrs .
physical locations of the est - ssrs were assigned by searching against the nonredundant ( nr ) protein database of ncbi ( http://www.ncbi.nlm.nih.gov/ ) and the brassica database ( http://brassicadb.org/brad/ ) using blastx .
our results showed that 4329 est - ssrs ( 44.4% ) were located in coding regions ( cdss ) , 3456 ( 35.5% ) in 5-utrs , and 1297 ( 13.3% ) in 3-utrs ( figure 4 , table s4 ) .
locations of the remaining 672 est - ssrs ( 6.9% ) were not successfully assigned ( figure 4 , table s4 ) .
for the est - ssrs localized in the cds region , trinucleotide repeats were the most common ones , accounting for 62.72% of the total est - ssrs localized in this region , followed by dinucleotide repeats ( 897 , 20.72% ) , mononucleotide repeats ( 325 , 7.51 ) , and compound formation ( 287 , 6.63% ) ( table s4 ) .
dinucleotide repeats ( 1909 , 55.24% ) were the dominant types in 5-utrs , followed by trinucleotide repeats ( 730 , 21.12% ) , mononucleotide repeats ( 483 , 13.98% ) , and compound formation ones ( 214 , 6.19% ) ( table s4 ) .
mono- , di- , and trinucleotide repeat est - ssrs were the top three types found in 3-utrs , accounting for 35.08% , 30.07% , and 28.60% of the total est - ssrs localized in these regions , respectively .
the est - ssrs and the flanking sequences ( 50  bp on each side ) were aligned to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn to determine their physical positions .
new est - ssrs were identified by comparing with the earlier reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .
a total of 2744 new est - ssrs ( 26.3% ) were identified in the study . of the 7676 known ssrs ( 73.6% ) , 2317 est - ssrs ( 22.2% ) show polymorphism with different repeat numbers , and 5359 ( 51.4% ) were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence   ( table s2 ) .
a total of 7877 pcr primer pairs from the unique sequences flanking 1561 est - ssr loci were designed according to the criteria described in section 2 using primer 3 ( table s5 ) . for each est - ssr locus , a maximum of 5 alternative primer pairs was designed .
the other 8859 est - ssrs , which had no appropriate pcr primer pairs designed as their flanking sequences , did not fulfill the primer design criteria mentioned above . for the 1561 est - ssrs with pcr primers designed ,
pcr primers of those aforementioned 24 loci with n  20  bp were selected for primer synthesis and amplification evaluation in chinese cabbage fushanbaotou .
nineteen ( 79.2% ) of these 24 est - ssr loci successfully yielded pcr amplicons in fushanbaotou .
we sequenced these nineteen pcr amplicons and found that the amplicons in thirteen loci were exactly the same as expected ; two were longer than the expected size , and four were shorter ( table 3 ) .
size deviation of five est - ssrs loci with the expected sizes ( br - es6 , br - es7 , br - es8 , br - es12 , and br - es18 ) was due to the variations of ssr repeat motifs ( table s6 ) .
one amplicon ( br - es16 ) deviated from the expected sizes and had an additional 86  bp containing a ( tc)9 motif near the ssr repeat motif region ( table s6 ) .
nineteen effective primer pairs were used for polymorphism validation for these aforementioned 24 chinese cabbage cultivars .
the results showed that 17 loci ( 89.5% ) were polymorphic ( figure 5 ) .
a total of 56 alleles at the 17 polymorphic loci were identified and the average number of alleles per ssr locus was 3.29 with a range between 2 and 6 .
a maximum of 6 alleles was detected for br - es16 and br - es18 loci .
br - es6 and br - es11 had no polymorphic allele in all 24 cultivars in this study ( figure 5 , tables 3 and s4 ) .
of the 17 polymorphic loci , twelve loci were polymorphic in all cultivars of b. pekinensis l. and b. chinensis l. three loci ( br - es2 , br - es9 , and br - es19 ) had no polymorphism in the cultivars of b. pekinensis l. but had polymorphism in the cultivars of b. chinensis l. , while two loci ( br - es4 and br - es7 ) were polymorphic in the cultivars of b. pekinensis l. but were not polymorphic in the cultivars of b. chinensis l. ( figure 5 , table s8 ) .
we sequenced the polymorphic alleles of the 17 polymorphic loci and found that polymorphisms of 9 loci ( br - es1 , br - es4 , br - es7 , br - es8 , br - es10 , br - es14 , br - es17 , br - es18 , and br - es19 ) were because of different iterate numbers of ssr repeat motifs . in another 6 polymorphic loci ( br - es2 , br - es3 , br - es12 , br - es13 , br - es15 , and br - es16 ) ,
the most polymorphic alleles were found in the repeat motifs with additional changes in other regions ( table s7 ) .
for example , compared with the allele br - es3 - 160  bp in fushanbaotou , the polymorphic alleles br - es3 - 163  bp and 145  bp had different iterate numbers of the tag / atc repeat motif , while the polymorphic allele 99  bp had not only a different number of the repeat motif , but also a deletion in another region ( table s7 ) .
the other two polymorphic loci , br - es5 and br - es9 , had polymorphisms that are not related with the repeat numbers of ssr motifs ( table s7 ) .
illumina paired - end rna sequencing is one of the fast immerging next - generation sequencing ( ngs ) technologies . because of its advantages in high - throughput , high accuracy , and low cost
, illumina paired - end sequencing has been widely used for de novo transcriptome sequencing and assembly and transcriptome quality and quantity analysis in many plants [ 37 , 38 , 42 , 43 ] . in our previous study ,
the transcriptome of rosette and folding leaves in chinese cabbage was analyzed using the illumina paired - end rna sequencing technology , and abundant clean reads and ests with high quality were obtained .
the large quantity of clean reads would increase coverage depth of transcriptome nucleotide , enhance sequencing accuracy , and provide useful information for developing new tools for genetic mapping and molecular breeding of chinese cabbage . in this study , we further assembled the clean reads into contigs and unigenes from the rl and fl libraries , respectively .
the parameters for both contigs and unigenes between the two libraries had no significant differences ( table 1 ) , indicating our illumina sequencing solutions have high reliability and reproducibility .
the unigenes of the two libraries were further assembled and a total of 51,694 nonredundant unigenes were obtained from the 40.7  mb sequence data .
we discovered more nonredundant unigenes than those in previous studies [ 35 , 36 ] , which represent a large portion of the chinese cabbage transcriptome and are important for a comprehensive understanding of est - ssrs .
a total of 10,420 ssrs with over 12  bp were identified from the deep transcriptome sequence dataset of chinese cabbage .
the frequency of occurrence of ssrs is slightly higher than those reported in previous studies on chinese cabbage ( about 8.415.6% ) [ 20 , 3436 ] and also higher than those of other dicotyledonous species such as peanut ( 6.8% ) , sweetpotato ( 8.2% ) , sesame ( 8.9% ) , pigeonpea ( 7.6% ) , grapes ( 2.5% ) , pepper ( 4.9% ) , and flax ( 3.5% ) , but it is lower than those of coffee ( 18.5% ) , radish ( 23.8% ) , and caster bean ( 28.4% ) .
detection of est - ssrs depends on a number of factors such as genome structure , tools and parameters for est - ssrs detection and exploration , and size of dataset for unigene assembly .
the frequency of ssrs with different sizes of repeat units is not evenly distributed in plants .
previous studies showed dinucleotide ssr loci are the most abundant class in safflower , pigeonpea , and sesame , whereas trinucleotide repeats are the most frequent ones in barley , sweetpotato , jatropha curcas , iris , pepper , caster bean , flax , cucurbita pepo , and radish . in ramie   and wheat , dinucleotide and trinucleotide
trinucleotide ( 4405 , 42.3% ) was found to be the most common repeat motif class in chinese cabbage , followed by dinucleotide ( 4043 , 38.8% ) ( figure 2 ) .
however , on the genomic level , of chinese cabbage , dinucleotide is the most common repeat motif , followed by trinucleotide .
we found the most dominant mononucleotide repeat motif in chinese cabbage was a / t ( 1,562 , accounting for 15.0% of the total est - ssrs ) , which is consistent with previous reports for chinese cabbage   and for other plants such as arabidopsis , rice , wheat , radish , castor bean , gossypium raimondii , oil palm , and eggplant .
for dinucleotide motif , ag / ct was the most common repeat motif , accounting for 87.0% of the total dinucleotide est - ssrs .
it is in close agreement with the results in previous studies for genic ssrs in chinese cabbage [ 20 , 36 ] and those in most other plants such as sweetpotato , iris , sesame , and radish .
the ag / ct repeat motif was also the most dominant repeat among all the est - ssrs identified in this study , accounting for 33.8% of the total est - ssrs .
however , for genomic - ssrs in chinese cabbage , at / ta is the most common dinucleotide motif .
the aag / ctt ( 1,445 , 13.9% ) motif was the most frequent motif among trinucleotide est - ssrs in the study , which is consistent with the results in previous studies in chinese cabbage [ 25 , 36 ] and many dicot species , for example , arabidopsis , soybean , peanut , sweetpotato , radish , and sesame .
in many monocot species such as maize , barley , and sorghum [ 66 , 67 ] , ccg / ggc is the most dominant trinucleotide repeat motif .
it is considered a specific feature of monocot genomes due to the high gc content in monocot genomes .
of all 10420 est - ssrs identified in this study , more than 70% have been identified and presented in the ssr marker database ( http://oilcrops.info/ssrdb ) , among which over half were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence ( table s2 ) .
2317 est - ssrs ( 22.2% ) with polymorphism in different repeat numbers could further be used for identification of chiifu-401 and fushanbaotou and for genetic linkage map constructions using these two cultivars as parents .
a total of 2744 new est - ssrs ( 26.3% ) were identified in the study , which , in combination with previously discovered est - ssrs , could be used for high - density genetic linkage map construction , gene / qtl mapping , cultivar identification , and so forth . in the present study ,
79.2% of the est - ssrs primer pairs selected for primer evaluation successfully generated high quality amplicons , indicating that the ests from the high - throughput rna sequencing of chinese cabbage transcriptome are suitable for specific primer design .
the unsuccessfully designed primer pairs may be due to splice sites , large introns , chimeric primer(s ) , or poor quality sequences .
we found that all amplicons contained the expected ssrs and the ssrs in 13 amplicons were exactly the same as predicted ( table s6 ) .
the deviation of est - ssr pcr amplicons from the expected size is likely due to the presence of introns , large insertions or repeat number variations , a lack of specificity , or assembly errors . in the present study
, we found five of six amplicons with unexpected sizes had different iterate number of ssr repeat units , while the other one had a 86  bp insertion near the expected ssr repeat motif region ( table s6 ) .
these results suggested that the unigenes assembled from the high - throughput rna sequencing of chinese cabbage transcriptome are reliable , and the est - ssrs identified in our dataset could be used for further studies , such as genetic mapping and cultivar identification .
most of the est - ssr loci ( accounting for 89.5% of the tested loci ) were found to be polymorphic among the 24 tested cabbage cultivars .
the mean number of alleles per ssr locus was 3.29 with a range between 2 and 6 ( table 3 ) , indicating that polymorphism of est - ssrs in chinese cabbage is relatively high .
most of the polymorphisms of the tested est - ssr loci are due to the variations of ssr repeat motifs in this study .
there were only two loci where the polymorphisms were not related to the ssr repeat motif variations ( table s6 ) .
the results indicate that the est - ssrs identified and the pcr primers designed in this study could further be used for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . in summary , we assembled a large set of clean reads with high quality derived from the chinese cabbage transcriptome using high - throughput rna sequencing technology with a solexa / illumina platform .
a total of 51,694 nonredundant unigenes were obtained from 40.7  mb sequence data , providing substantial knowledge for est - ssr identification and characterization .
10,420 est - ssrs were identified and characterized , and pcr primer pairs for 1561 est - ssrs were designed . by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) , we identified a total of 2744 new est - ssrs .
primer pairs for 24 est - ssrs were selected for primer evaluation , and 79.2% of the 24 est - ssr loci successfully generated high quality amplicons . among the effective primers , 89.5% of them showed polymorphism in 24 cultivars of chinese cabbage .
the est - ssrs developed in this study , in combination with previously reported est - ssrs , will provide valuable resources for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . to our knowledge , this is the first successful attempt to develop large quantity of est - ssrs with high quality based on the transcriptome of chinese cabbage using high - throughput rna sequencing technology .
 simple sequence repeats ( ssrs ) are among the most important markers for population analysis and have been widely used in plant genetic mapping and molecular breeding . expressed sequence tag - ssr ( est - ssr ) markers , located in the coding regions , are potentially more efficient for qtl mapping , gene targeting , and marker - assisted breeding . in this study 
 , we investigated 51,694 nonredundant unigenes , assembled from clean reads from deep transcriptome sequencing with a solexa / illumina platform , for identification and development of est - ssrs in chinese cabbage . in total , 10,420 est - ssrs with over 12  bp were identified and characterized , among which 2744 est - ssrs are new and 2317 are known ones showing polymorphism with previously reported ssrs . 
 a total of 7877 pcr primer pairs for 1561 est - ssr loci were designed , and primer pairs for twenty - four est - ssrs were selected for primer evaluation . in nineteen est - ssr loci ( 79.2% ) , amplicons were successfully generated with high quality . 
 seventeen ( 89.5% ) showed polymorphism in twenty - four cultivars of chinese cabbage . 
 the polymorphic alleles of each polymorphic locus were sequenced , and the results showed that most polymorphisms were due to variations of ssr repeat motifs . 
 the est - ssrs identified and characterized in this study have important implications for developing new tools for genetics and molecular breeding in chinese cabbage . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion