Research Article Characterization and Development of EST-SSRs …downloads.hindawi.com/journals/ijg/2015/473028.pdf · 2019-07-31 · Research Article Characterization and Development

Research ArticleCharacterization and Development of EST-SSRs byDeep Transcriptome Sequencing in Chinese Cabbage(Brassica rapa L ssp pekinensis)

Qian Ding1 Jingjuan Li2 Fengde Wang2 Yihui Zhang2 Huayin Li2

Jiannong Zhang1 and Jianwei Gao2

1College of Horticulture Gansu Agricultural University Lanzhou 730070 China2Institute of Vegetables and Flowers Shandong Academy of Agricultural Sciences and Shandong Key Laboratory ofGreenhouse Vegetable Biology and Shandong Branch of National Vegetable Improvement Center Jinan 250100 China

Correspondence should be addressed to Jiannong Zhang zjn0101sinacom and Jianwei Gao jianweigao3yahoocom

Received 15 February 2015 Accepted 26 March 2015

Academic Editor Yanbin Yin

Copyright copy 2015 Qian Ding et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Simple sequence repeats (SSRs) are among the most important markers for population analysis and have been widely used inplant genetic mapping and molecular breeding Expressed sequence tag-SSR (EST-SSR) markers located in the coding regionsare potentially more efficient for QTL mapping gene targeting and marker-assisted breeding In this study we investigated 51694nonredundant unigenes assembled from clean reads from deep transcriptome sequencing with a SolexaIllumina platform foridentification and development of EST-SSRs in Chinese cabbage In total 10420 EST-SSRs with over 12 bp were identified andcharacterized among which 2744 EST-SSRs are new and 2317 are known ones showing polymorphism with previously reportedSSRsA total of 7877PCRprimer pairs for 1561 EST-SSR lociwere designed andprimer pairs for twenty-four EST-SSRswere selectedfor primer evaluation In nineteenEST-SSR loci (792) ampliconswere successfully generatedwith high quality Seventeen (895)showed polymorphism in twenty-four cultivars of Chinese cabbage The polymorphic alleles of each polymorphic locus weresequenced and the results showed that most polymorphisms were due to variations of SSR repeat motifs The EST-SSRs identifiedand characterized in this study have important implications for developing new tools for genetics andmolecular breeding inChinesecabbage

1 Introduction

Chinese cabbage (Brassica rapa L ssp pekinensis) is a diploid(2119899 = 2119909 = 20) dicot with a genomic size of 550Mb(httpwwwbrassicainforesource) It is a subspecies of Brapa with the A genome [1] The species originated in Chinaand now has become one of the most important and widelycultivated leaf vegetables in Asia Chinese cabbage has rosetteleaves (RLs) and folding leaves (FLs) The tight leafy head isthe main edible part After a long history of domesticationChinese cabbage evolves into different cultivars with a varietyof characteristics such as rosette leaf morphology headingleaf morphology leafy head shape size and structure flow-ering time nutrient composition and resistance to biotic andabiotic A better understanding of the molecular mechanismof evolution of Chinese cabbage and further development of

marker-assisted selection (MAS) will accelerate the selectionprocess of improved cultivars tomeet the growing consumersand environmental needs Although progress has been madein underlining themolecularmechanism [2ndash5]many aspectsare still unclear

Molecular markers have been widely used to study thegenetic basis of important traits and map regulatory genesin plants Markers tightly linked with important agronomictraits can potentially be used for molecular breeding todevelop improved cultivars Many molecular markers andgenetic maps of Chinese cabbage have been reported previ-ously [6ndash25] However there is still a great need to developnovel molecular markers for construction of high-densitylinkage maps for genetics andmolecular studies of importanttraits in Chinese cabbage

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2015 Article ID 473028 11 pageshttpdxdoiorg1011552015473028

2 International Journal of Genomics

Simple sequence repeat (SSR) markers or microsatel-lite markers are among the most important markers inplants SSRs have been widely used in genetic mappingand molecular breeding in plants because they are highlyabundant and have significant polymorphism Other factorslike accessibility for detection reliability and codominancealso make them perfect markers for such purposes [26]SSRs found in transcribed sequences are called expressedsequence-simple sequence repeats (EST-SSRs) Comparedwith genomic-SSRs detected in noncoding sequences EST-SSRs are more efficient for QTL mapping gene targetingand MAS [27] As transcribed sequences are more conservedthan noncoding sequences the transferability of EST-SSRsis better than genomic-SSRs [28ndash30] which can be utilizedfor cross genome comparison and evolutionary analysis [2731] Additionally abundant ESTs were generated in recentyears with the development of next-generation sequencingapproaches making identification of EST-SSRs more practi-cal and cost-efficient [32] Many EST-SSRs have been iden-tified in Chinese cabbage [16 20 25 33ndash36] Because wholegenome sequencing of Chinese cabbage is still underway newEST-SSRs could also be identified for further studies suchas high-density genetic linkage map construction geneQTLmapping and cultivar identification

In our previous study the whole transcriptomes wereanalyzed for the rosette leaves and folding leaves of atypical heading Chinese cabbage namely FuShanBaoTouusing a SolexaIllumina RNA-Seq platform and a large-scaleEST database was generated [37] In this study we furtherassembled those ESTs from the RL and FL libraries intononredundant unigenes A total of 10420 EST-SSRs wereidentified among which 2744 EST-SSRs are detected forthe first time according to the SSR marker database forBrassica (httpoilcropsinfoSSRdb) We characterized theseidentified EST-SSRs and designed 7877 PCR primer pairs for1561 EST-SSRs Furthermore serving as a validation purposewe tested polymorphisms of 24 EST-SSRs We expect thisstudy can pave the road for further investigation of new EST-SSR markers and for construction of high-density geneticmaps

2 Materials and Methods

21 Plant Materials For EST-SSR identification and primerdesign a typical heading Chinese cabbage namely FuShan-BaoTou was used in this study For primer assessmentand SSR polymorphism analysis a panel of twenty-fourcultivars of Chinese cabbage was used including nineteenmorphologically diverse cultivars of Brassica rapa L ssppekinensis (B pekinensis L) and five Brassica rapa L chinensis(B chinensis L) All plants were grown in a greenhouse with168 photoperiod at 22plusmn2∘C Leaves were collected after theywere grown for two weeks from ten seedlings of each cultivarand were pooled together for DNA extraction

22 De Novo Assembly We assembled the clean read datasetpresented by Wang et al [37] from the RL and FL librariesaccording to the methods described byWang et al [38] usingthe Trinity software (httptrinityrnaseqsourceforgenet)

Contigs and unigenes were obtained from these two librariesrespectively Redundant sequences were removed and over-lapping unigenes were assembled into continuous sequencesby the TIGR Gene Indices Clustering (TGICL) tools [39]Similarity was set at 94 and an overlap length was set at100 bp

23 Identification of EST-Derived SSRs and Primer DesignSSRs were detected with the MicroSAtellite software (MISAhttppgrcipk-gaterslebendemisa) Parameters were setwith a minimum number of 12 6 5 5 4 and 4 repeatunits for identification of mono- di- tri- tetra- penta- andhexanucleotide motifs respectively Primers were designedusing primer 3 with no SSR allowed in primers Primer lengthranged from 18 to 28 bp (with an optimality at 23) Annealingtemperature was set at 55ndash65∘C (with an optimality at 60∘C)The size of a PCR product ranged from 80 to 300 bp

24 Mapping EST-SSRs The physical positions of theEST-SSRs identified in the study were determined byaligning the SSRs and flanking sequences (50 bp at eachside) to the Brassica rapa (Chiifu-401) reference genome(httpbrassicadborgbrad) using BLASTN New EST-SSRs were identified by comparing with previouslyreported SSRs in the SSR marker database for Brassica(httpoilcropsinfoSSRdb) [25]

25 SSRAmplification and SSR PolymorphismAnalysis DNAwas extracted following a CTAB DNA extraction protocol[40] The DNA sample of the Chinese cabbage FuShan-BaoTou was used as template to detect the availability ofSSR primers designed above The DNA samples of thoseaforementioned twenty-four cultivars of Chinese cabbagewere used as templates for SSR polymorphism analysis Thepolymorphisms of EST-SSRs were validated by 6 denatur-ing polyacrylamide gel electrophoresis 12 nondenaturingpolyacrylamide gel electrophoresis and sequencing

3 Results

31 De Novo Assembly High quality clean read data fromthe RL and FL libraries by Wang et al [37] were assembledusing the Trinity software package [41] A total of 99684 and95411 contigs were obtained with an average length of 333and 342 bp and amedian length (N50) of 531 and 536 bp fromthe RL and FL libraries respectively (Table 1)

Contigs from the same transcript were detected withpaired-end reads as well as the distances between thesecontigs Using the Trinity software package we assembledthese contigs into unigenes in whichNswere removedTheseunigenes were set to be not extendable on either end of thesequences A total of 46294 and 48473 unigenes from theRL and FL libraries were obtained with an average lengthof 707 and 680 bp and a median length (N50) of 1000 and980 bp respectively (Table 1) Size distribution of the contigsand unigenes is consistent with the RL and FL libraries asshown in Figure 1 indicating that our Illumina sequencingsolution is reliable and reproducible Unigenes from the twosamples were combined redundant unigenes were removed

International Journal of Genomics 3

Table 1 Overview of the sequencing and assembly

Sample Total number Total length (nt) Average length(nt) N50 Total consensus

sequences Distinct clusters Distinctsingletons

Contig RL 99684 33205708 333 531 mdash mdash mdashFL 95441 32596297 342 536 mdash mdash mdash

UnigeneRL 46294 32729586 707 1000 46294 19512 26782FL 48473 32971187 680 980 48473 19749 28724All 51694 40724256 788 1154 51694 23850 27844

RLFL

1

10

100

1000

10000

RLFL

All unigenes

1

10

100

1000

10000

100000

100000

Contig

Unigene

Num

ber o

f rea

ds

Num

ber o

f rea

ds

Sequence size (bp)

Sequence size (bp)

le200

201

ndash300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

140

1ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000

le300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

1401

ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000

Figure 1 Size distribution of the assembled contigs and unigenes in RL and FL libraries

and the rest was assembled with TGICL [39] to form a singledataset which represents 407Mb of sequence and containsa total of 51694 nonredundant unigenes with an averageread length of 788 bp and a median read length (N50) of1154 bp (Table 1) The sequences of the unigenes are listedin Table s1 (see Supplementary Material available online athttpdxdoiorg1011552015473028)

The length of 24271 nonredundant unigenes (4695)is between 200 and 500 bp the length of 13613 (2633) isbetween 501 and 1000 bp and the length of 13810 (2672) islonger than 1000 bp (Figure 1)

32 Characterization of EST-SSRs in Chinese Cabbage Atotal of 10420 EST-SSRs were detected with the MicroSAtel-lite software (MISA httppgrcipk-gaterslebendemisa) in8571 unigenes accounting for 166 of total nonredundantunigenes (Tables 2 and s2) The mean SSR density is oneper 39 Kb corresponding to one for every 50 nonredundantunigenes 1502 unigenes (175) harbored more than one SSRand 666 SSRs (64) were present in compound formationthat had more than one repeat type (Table 2)

The size of SSR repeat units ranged from one to sixThe number of SSRs with each repeat unit was found to be


Table 2 Summary of EST-SSR searching results

Searching items NumbersTotal number of sequences examined 51694Total size of examined sequences (bp) 40724256Total number of identified SSRs 10420Number of SSR-containing sequences 8571Number of sequences containing more than one SSR 1502Number of SSRs present in compound formation 666 EST-SSRs 166

1644

4043

4405

90 112 1260

1000

2000

3000

4000

5000

Num

ber o

f EST

-SSR

s

Mon

onuc

leot

ide

Din

ucle

otid

e

Trin

ucle

otid

e

Tetr

anuc

leot

ide

Pent

anuc

leot

ide

Hex

anuc

leot

ide

Figure 2 EST-SSR statistics

quite different The SSRs with tri- and dinucleotide repeatmotifs were themost common (4405 4227 4043 3880resp) followed by mono- (1644 1578) hexa- (126 121)penta- (112 107) and tetra- (90 086) nucleotide repeatmotifs (Figure 2) The most common two repeat motif typesaccounted for 8107 of the total SSRs detected and the restrepeat motifs types only accounted for 1893

The iterate number of repeat units in an EST-SSR rangedfrom 4 to 25 The occurrence frequency of EST-SSTs withdifferent iterate numberswas found to be unequal either EST-SSRs with iterate number of 5 (2832 2718) were the mostcommon ones followed by 6 (2739 2629) 7 (1368 1313)8 (703 675) 12 (542 520) and 9 (480 461) (Tables3) A dinucleotide containing EST-SSRs with a maximum of25 repeat units was identified For EST-SSRs with more than10 repeat units the mononucleotide repeat motifs were themost abundant accounting for 9346 of these EST-SSRsThe 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-SSRsare from 12 to 20 bp accounting for 9147 of the total EST-SSRs followed by EST-SSRs with 21ndash30 bp in length (874SSRs 839) Only 13 EST-SSRs were identified with over30 bp accounting for 012 of the total EST-SSRs

A total of 124 EST-SSR motifs were identified including2 mono- 3 di- 10 tri- 13 tetra- 33 penta- and 63 hex-anucleotide repeat units containing EST-SSRsThe dominantmotif identified in our EST-SSRs was AGCT (3519 338)followed by AT (1562 150) AAGCTT (1445 139)AGGCCT (776 74) ATCATG (627 60) AACGTT(392 44) ACCGGT (392 38) ACGT (349 33) andAGCCTG (317 30) (Figure 3) The other 115 motifs havelow frequency accounting only for 93 of total EST-SSRs

Physical locations of the EST-SSRs were assigned bysearching against the nonredundant (nr) protein databaseof NCBI (httpwwwncbinlmnihgov) and the Brassicadatabase (httpbrassicadborgbrad) using BLASTX Ourresults showed that 4329 EST-SSRs (444) were locatedin coding regions (CDSs) 3456 (355) in 51015840-UTRs and1297 (133) in 31015840-UTRs (Figure 4 Table s4) Locations ofthe remaining 672 EST-SSRs (69) were not successfullyassigned (Figure 4 Table s4) For the EST-SSRs localizedin the CDS region trinucleotide repeats were the mostcommon ones accounting for 6272 of the total EST-SSRslocalized in this region followed by dinucleotide repeats (8972072) mononucleotide repeats (325 751) and compoundformation (287 663) (Table s4) Dinucleotide repeats (19095524) were the dominant types in 51015840-UTRs followed bytrinucleotide repeats (730 2112) mononucleotide repeats(483 1398) and compound formation ones (214 619)(Table s4) Mono- di- and trinucleotide repeat EST-SSRswere the top three types found in 31015840-UTRs accounting for3508 3007 and 2860 of the total EST-SSRs localizedin these regions respectively

33 New EST-SSRs Identification The EST-SSRs and theflanking sequences (50 bp on each side) were alignedto the Brassica rapa (Chiifu-401) reference genome(httpbrassicadborgbrad) using BLASTN to determinetheir physical positions New EST-SSRs were identified bycomparing with the earlier reported SSRs in the SSR markerdatabase for Brassica (httpoilcropsinfoSSRdb) A totalof 2744 new EST-SSRs (263) were identified in the studyOf the 7676 known SSRs (736) 2317 EST-SSRs (222)show polymorphismwith different repeat numbers and 5359(514) were exactly the same with the earlier reported SSRsbased on the Brassica rapa (Chiifu-401) genomic sequence[25] (Table s2)

34 Primer Design and Evaluation of EST-SSRs in ChineseCabbage A total of 7877 PCR primer pairs from the uniquesequences flanking 1561 EST-SSR loci were designed accord-ing to the criteria described in Section 2 using primer 3 (Tables5) For each EST-SSR locus a maximum of 5 alternativeprimer pairs was designed The other 8859 EST-SSRs whichhad no appropriate PCR primer pairs designed as theirflanking sequences did not fulfill the primer design criteria


Table 3 Details of 19 EST-SSRs that successfully yielded PCR amplicons in FuShanBaoTou

Code EST-SSR name Motif Product size expected (bp) Product size validated (bp) SSR location Number of allelesBR-es1 CL3455Contig1 All-2 (TC)11 97 97 CDS 2BR-es2 CL4114Contig2 All-2 (TCA)7 157 157 3-UTR 3BR-es3 CL2525Contig4 All-1 (TAG)9 160 160 CDS 4BR-es4 Unigene10387 All-1 (CTC)9 127 127 CDS 2BR-es5 CL7077Contig2 All-1 (AATC)5 153 153 3-UTR 3BR-es6 Unigene16359 All-1 (AACC)5 134 130 5-UTR 1BR-es7 CL4685Contig1 All-1 (CCTT)6 160 148 3-UTR 2BR-es8 CL5247Contig3 All-1 (TTTC)6 133 141 CDS 3BR-es9 CL3462Contig4 All-1 (AATCG)4 155 155 CDS 2BR-es10 CL5726Contig2 All-2 (TCTCT)4 146 146 5-UTR 3BR-es11 Unigene6713 All-1 (AAAAC)4 118 118 CDS 1BR-es12 CL7282Contig2 All-1 (GAGGA)5 140 117 CDS 4BR-es13 Unigene2970 All-1 (GAACT)5 106 106 3-UTR 3BR-es14 Unigene8739 All-1 (GATTT)5 130 130 5-UTR 4BR-es15 CL5873Contig4 All-2 (CCCTAA)4 146 146 3-UTR 4BR-es16 Unigene14449 All-1 (CTCAAG)5 99 185 CDS 6BR-es17 Unigene5096 All-1 (ACTCCC)5 141 141 CDS 3BR-es18 CL4691Contig2 All-1 (GATGGT)7 155 117 CDS 6BR-es19 Unigene13507 All-1 (ATTTG)4 152 152 CDS 2EST-SSRs shown in bold have sizes different from the expected sizes

150

08 33

338

17 44

139

08 38

08 08 30

74 60

13 31

00

50

100

150

200

250

300

350

400

Freq

uenc

y (

)

Motif

AT

CG

ACG

T

AGC

T

ATA

T

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

T

ACG

CG

T

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

G

Oth

ers

Figure 3 Frequency distribution of EST-SSRs according to motif sequence types

mentioned above For the 1561 EST-SSRs with PCR primersdesigned PCR primers of those aforementioned 24 loci with119899 ge 20 bpwere selected for primer synthesis and amplificationevaluation in Chinese cabbage FuShanBaoTou Nineteen(792) of these 24 EST-SSR loci successfully yielded PCRamplicons in FuShanBaoTou We sequenced these nineteenPCR amplicons and found that the amplicons in thirteen lociwere exactly the same as expected two were longer than theexpected size and four were shorter (Table 3) Size deviationof five EST-SSRs loci with the expected sizes (BR-es6 BR-es7 BR-es8 BR-es12 and BR-es18) was due to the variationsof SSR repeat motifs (Table s6) One amplicon (BR-es16)

deviated from the expected sizes and had an additional 86 bpcontaining a (TC)

9motif near the SSR repeat motif region

(Table s6)

35 Validation of Polymorphism of EST-SSRs Nineteen effec-tive primer pairs were used for polymorphism validationfor these aforementioned 24 Chinese cabbage cultivarsThe results showed that 17 loci (895) were polymorphic(Figure 5) A total of 56 alleles at the 17 polymorphic loci wereidentified and the average number of alleles per SSR locuswas 329 with a range between 2 and 6 A maximum of 6alleles was detected for BR-es16 and BR-es18 loci BR-es6 and


355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR

Figure 4 Frequency distribution of EST-SSRs based on locations

BR-es11 had no polymorphic allele in all 24 cultivars in thisstudy (Figure 5 Tables 3 and s4) Of the 17 polymorphic locitwelve loci were polymorphic in all cultivars of B pekinensisL and B chinensis L Three loci (BR-es2 BR-es9 and BR-es19) had no polymorphism in the cultivars of B pekinensisL but had polymorphism in the cultivars of B chinensis Lwhile two loci (BR-es4 and BR-es7) were polymorphic in thecultivars of B pekinensis L but were not polymorphic in thecultivars of B chinensis L (Figure 5 Table s8)

We sequenced the polymorphic alleles of the 17 polymor-phic loci and found that polymorphisms of 9 loci (BR-es1BR-es4 BR-es7 BR-es8 BR-es10 BR-es14 BR-es17 BR-es18and BR-es19) were because of different iterate numbers ofSSR repeat motifs In another 6 polymorphic loci (BR-es2BR-es3 BR-es12 BR-es13 BR-es15 and BR-es16) the mostpolymorphic alleles were found in the repeat motifs withadditional changes in other regions (Table s7) For examplecompared with the allele BR-es3-160 bp in FuShanBaoTouthe polymorphic alleles BR-es3-163 bp and 145 bp had differ-ent iterate numbers of the TAGATC repeat motif while thepolymorphic allele 99 bp had not only a different number ofthe repeat motif but also a deletion in another region (Tables7) The other two polymorphic loci BR-es5 and BR-es9 hadpolymorphisms that are not related with the repeat numbersof SSR motifs (Table s7)

4 Discussion

41 High-Throughput RNA Sequencing Provides SubstantialKnowledge for EST-SSRs Illumina paired-endRNA sequenc-ing 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-endsequencing has been widely used for de novo transcriptomesequencing and assembly and transcriptome quality andquantity analysis in many plants [37 38 42 43] In our

previous study the transcriptome of rosette and foldingleaves in Chinese cabbage was analyzed using the Illuminapaired-end RNA sequencing technology and abundant cleanreads and ESTs with high quality were obtained [37] Thelarge quantity of clean reads would increase coverage depthof transcriptome nucleotide enhance sequencing accuracyand provide useful information for developing new toolsfor genetic mapping and molecular breeding of Chinesecabbage In this study we further assembled the clean readsinto contigs and unigenes from the RL and FL librariesrespectively The parameters for both contigs and unigenesbetween the two libraries had no significant differences(Table 1) indicating our Illumina sequencing solutions havehigh reliability and reproducibility The unigenes of thetwo libraries were further assembled and a total of 51694nonredundant unigenes were obtained from the 407Mbsequence data We discovered more nonredundant unigenesthan those in previous studies [35 36] which represent alarge portion of the Chinese cabbage transcriptome and areimportant for a comprehensive understanding of EST-SSRs

42 Frequency and Distribution of EST-SSRs in ChineseCabbage A total of 10420 SSRs with over 12 bp wereidentified from the deep transcriptome sequence dataset ofChinese cabbage About 166 of the unigenes have SSRsThe frequency of occurrence of SSRs is slightly higherthan those reported in previous studies on Chinese cabbage(about 84ndash156) [20 34ndash36] and also higher than thoseof other dicotyledonous species such as peanut (68) [44]sweetpotato (82) [21] sesame (89) [43] pigeonpea (76)[45] grapes (25) [46] pepper (49) [47] and flax (35)[48] but it is lower than those of coffee (185) [49] radish(238) [38] and caster bean (284) [50] Detection ofEST-SSRs depends on a number of factors such as genomestructure [51] tools and parameters for EST-SSRs detectionand exploration [43] and size of dataset for unigene assembly[27]

The frequency of SSRs with different sizes of repeat unitsis not evenly distributed in plants Previous studies showeddinucleotide SSR loci are themost abundant class in safflower[52] pigeonpea [45] and sesame [43] whereas trinucleotiderepeats are the most frequent ones in barley [53] sweetpotato[21] Jatropha curcas [54] iris [55] pepper [47] caster bean[50] flax [48] Cucurbita pepo [56] and radish [38] In ramie[57] and wheat [58] dinucleotide and trinucleotide repeatmotifs are the most two abundant types In the presentstudy trinucleotide (4405 423) was found to be the mostcommon repeat motif class in Chinese cabbage followed bydinucleotide (4043 388) (Figure 2) It is consistent withprevious reports for SSRs identification from unigenes ofChinese cabbage [20] However on the genomic level ofChinese cabbage dinucleotide is the most common repeatmotif followed by trinucleotide [25]

We found the most dominant mononucleotide repeatmotif in Chinese cabbage was AT (1562 accounting for150 of the total EST-SSRs) which is consistent with pre-vious reports for Chinese cabbage [25] and for other plantssuch as Arabidopsis [59] rice [59] wheat [60] radish [38]castor bean [50] Gossypium raimondii [61] oil palm [62]


BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp

BR-es13 105 bp104 bp

101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp

BR-es2151bp157bp165bp

BR-es15158bp152bp 146bp

134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp

BR-es11 118 bpBR-es10

133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Figure 5 PCR products amplified by nineteen effective EST-SSR primer pairs in twenty-four cultivars of Chinese cabbage The order ofDNA samples from lane 1 to lane 24 within each primer pair image panel is 682 GuangDongZao ZaoHuangBai Z61-8 FuShanBaoTouLi-3 212-7 TianJinQingMaYe KuaiCai number 6-5 JinHuangXiaoBaiCai SiJiXiaoBaiCai SiJiHuangYangXiaoBaiCai PinZao number 1HanYuTeXuanHuangXin QuanNengSiJiKuaiCai JingYouXiaoBaiCaiKuaiCai GaoLiWaWaCai KeYiXiaWaWa JinNuoChunQiuWaWaCaiSiJiLvGanXiaoKuCai YouLv157 ShuYaoYouCai DeGaoYouLiangQingGengCai and QingXiuF1QingGengCai PCR products amplifiedby BR-es2 BR-es3 BR-es6 BR-es7 BR-es12 BR-es13 BR-es14 BR-es16 and BR-es19 primer pairs were separated on 6 denaturingpolyacrylamide gels while those amplified by BR-es1 BR-es4 BR-es5 BR-es8 BR-es9 BR-es10 BR-es11 BR-es15 BR-es17 and BR-es18 primerpairs were separated on 12 nondenaturing polyacrylamide gels

and eggplant [63] For dinucleotide motif AGCT was themost common repeat motif accounting for 870 of thetotal dinucleotide EST-SSRs It is in close agreement withthe results in previous studies for genic SSRs in Chinesecabbage [20 36] and those in most other plants such assweetpotato [21] iris [55] sesame [43] and radish [38] TheAGCT repeat motif was also the most dominant repeatamong all the EST-SSRs identified in this study accounting

for 338 of the total EST-SSRs However for genomic-SSRsin Chinese cabbage ATTA is themost common dinucleotidemotif [25] The AAGCTT (1445 139) motif was the mostfrequent motif among trinucleotide EST-SSRs in the studywhich is consistent with the results in previous studies inChinese cabbage [25 36] andmanydicot species for exampleArabidopsis [64] soybean [65] peanut [44] sweetpotato [21]radish [38] and sesame [43] In many monocot species such


as maize barley and sorghum [66 67] CCGGGC is themost dominant trinucleotide repeat motif It is considereda specific feature of monocot genomes due to the high GCcontent in monocot genomes [68]

43 New EST-SSRs Identification Of all 10420 EST-SSRs identified in this study more than 70 have beenidentified and presented in the SSR marker database(httpoilcropsinfoSSRdb) among which over half wereexactly the same with the earlier reported SSRs based onthe Brassica rapa (Chiifu-401) genomic sequence (Tables2) [25] It demonstrates that our method is highly reliablefor EST-SSR identification 2317 EST-SSRs (222) withpolymorphism in different repeat numbers could furtherbe used for identification of Chiifu-401 and FuShanBaoTouand for genetic linkage map constructions using thesetwo cultivars as parents A total of 2744 new EST-SSRs(263) were identified in the study which in combinationwith previously discovered EST-SSRs could be used forhigh-density genetic linkage map construction geneQTLmapping cultivar identification and so forth

44 High Polymorphism of Chinese Cabbage EST-SSRs In thepresent study 792 of the EST-SSRs primer pairs selected forprimer evaluation successfully generated high quality ampli-cons indicating that the ESTs from the high-throughput RNAsequencing of Chinese cabbage transcriptome are suitable forspecific primer design The unsuccessfully designed primerpairs may be due to splice sites large introns chimericprimer(s) or poor quality sequences [27] We sequenced allPCR amplicons in Chinese cabbage FuShanBaoTou yielding19 successful primer pairs We found that all ampliconscontained the expected SSRs and the SSRs in 13 ampliconswere exactly the same as predicted (Table s6)Thedeviation ofEST-SSR PCR amplicons from the expected size is likely dueto the presence of introns large insertions or repeat numbervariations a lack of specificity or assembly errors [43] In thepresent study we found five of six amplicons with unexpectedsizes had different iterate number of SSR repeat units whilethe other one had a 86 bp insertion near the expected SSRrepeat motif region (Table s6) These results suggested thatthe unigenes assembled from the high-throughput RNAsequencing of Chinese cabbage transcriptome are reliableand the EST-SSRs identified in our dataset could be usedfor further studies such as genetic mapping and cultivaridentification

Most of the EST-SSR loci (accounting for 895 of thetested loci) were found to be polymorphic among the 24tested cabbage cultivars The mean number of alleles perSSR locus was 329 with a range between 2 and 6 (Table 3)indicating that polymorphism of EST-SSRs in Chinese cab-bage is relatively high Most of the polymorphisms of thetested EST-SSR loci are due to the variations of SSR repeatmotifs in this study There were only two loci where thepolymorphisms were not related to the SSR repeat motifvariations (Table s6) The results indicate that the EST-SSRsidentified and the PCR primers designed in this study couldfurther be used for constructing high-density genetic linkagemaps mapping quantitative trait loci assessing germplasm

polymorphism and evolution marker-assisted selection andcloning functional gene in Chinese cabbage

In summary we assembled a large set of clean reads withhigh quality derived from the Chinese cabbage transcrip-tome using high-throughput RNA sequencing technologywith a SolexaIllumina platform A total of 51694 nonre-dundant unigenes were obtained from 407Mb sequencedata providing substantial knowledge for EST-SSR identi-fication and characterization 10420 EST-SSRs were iden-tified and characterized and PCR primer pairs for 1561EST-SSRs were designed By comparing with previouslyreported SSRs in the SSR marker database for Brassica(httpoilcropsinfoSSRdb) we identified a total of 2744new EST-SSRs Primer pairs for 24 EST-SSRs were selectedfor primer evaluation and 792 of the 24 EST-SSR locisuccessfully generated high quality amplicons Among theeffective primers 895 of them showed polymorphism in24 cultivars of Chinese cabbage The EST-SSRs developedin this study in combination with previously reported EST-SSRs will provide valuable resources for constructing high-density genetic linkage maps mapping quantitative trait lociassessing germplasm polymorphism and evolution marker-assisted selection and cloning functional gene in Chinesecabbage To our knowledge this is the first successful attemptto develop large quantity of EST-SSRs with high qualitybased on the transcriptome of Chinese cabbage using high-throughput RNA sequencing technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Qian Ding and Jingjuan Li contributed equally to this work

Acknowledgments

This research was supported by the China PostdoctoralScience Foundation Funded Project (2013M541948) theShandong Postdoctoral Science Foundation Funded ProjectChina (201303030) the National Natural Science Foundationof China (31401869) the National High-tech RampD Pro-gram of China (863 Program) (Grant 2012AA100103) theModern Agricultural Industrial Technology System Fundingof Shandong Province China (SDAIT-02-022-04) and theProject for Cultivation ofMajor Achievements in Science andTechnology in SAAS (2015CGPY09)

References

[1] X Wang H Wang J Wang et al ldquoThe genome of themesopolyploid crop species Brassicarapardquo Nature Genetics vol43 pp 1035ndash1039 2011

[2] X Yu J Peng X Feng et al ldquoCloning and structural andexpressional characterization of BcpLH gene preferentiallyexpressed in folding leaf of Chinese cabbagerdquo Science in ChinaSeries C Life Sciences vol 43 no 3 pp 328ndash329 2000


[3] H Wu L Yu X-R Tang R-J Shen and Y-K He ldquoLeafdownward curvature and delayed flowering caused by AtLHoverexpression in Arabidopsis thalianardquo Acta Botanica Sinicavol 46 no 9 pp 1106ndash1113 2004

[4] J Lee C T Han and Y Hur ldquoOverexpression of BrMORN anovel lsquomembrane occupation and recognition nexusrsquo motif pro-tein gene from Chinese cabbage promotes vegetative growthand seed production in Arabidopsisrdquo Molecules and Cells vol29 no 2 pp 113ndash122 2010

[5] B Wang X Zhou F Xu and J Gao ldquoEctopic expression ofa Chinese cabbage BrARGOS gene in Arabidopsis increasesorgan sizerdquo Transgenic Research vol 19 no 3 pp 461ndash472 2010

[6] KM Song J Y Suzuki M K Slocum PMWilliams and T COsborn ldquoA linkagemap of Brassica rapa (syn campestris) basedon restriction fragment length polymorphism locirdquoTheoreticaland Applied Genetics vol 82 no 3 pp 296ndash304 1991

[7] Y-S Chyi M E Hoenecke and J L Sernyk ldquoA genetic linkagemap of restriction fragment length polymorphism loci forBrassica rapa (syn campestris)rdquoGenome vol 35 no 5 pp 746ndash757 1992

[8] K Suwabe H Iketani T Nunome T Kage and M HiraildquoIsolation and characterization of microsatellites in Brassicarapa Lrdquo Theoretical and Applied Genetics vol 104 no 6-7 pp1092ndash1098 2002

[9] A J Lowe C Moule M Trick and K J Edwards ldquoEfficientlarge-scale development of microsatellites for marker andmapping applications in Brassica crop speciesrdquo Theoretical andApplied Genetics vol 108 no 6 pp 1103ndash1112 2004

[10] K Suwabe H Iketani T Nunome A Ohyama M Hiraiand H Fukuoka ldquoCharacteristics of microsatellites in Brassicarapa genome and their potential utilization for comparativegenomics in cruciferaerdquo Breeding Science vol 54 no 2 pp 85ndash90 2004

[11] S R Choi G R Teakle P Plaha et al ldquoThe referencegenetic linkagemap for themultinational Brassica rapa genomesequencing projectrdquo Theoretical and Applied Genetics vol 115no 6 pp 777ndash792 2007

[12] S K Jung Y C Tae G J King et al ldquoA sequence-tagged linkagemap of Brassica rapardquo Genetics vol 174 no 1 pp 29ndash39 2006

[13] K Suwabe H Tsukazaki H Iketani et al ldquoSimple sequencerepeat-based comparative genomics between Brassica rapa andArabidopsis thaliana the genetic origin of clubroot resistancerdquoGenetics vol 173 no 1 pp 309ndash319 2006

[14] J Wu Y-X Yuan X-W Zhang et al ldquoMapping QTLs formineral accumulation and shoot dry biomass under differentZn nutritional conditions in Chinese cabbage (Brassica rapa Lssp pekinensis)rdquo Plant and Soil vol 310 no 1-2 pp 25ndash40 2008

[15] F Li H Kitashiba K Inaba and T Nishio ldquoA brassica rapalinkage map of EST-based SNP markers for identification ofcandidate genes controlling flowering time and leafmorpholog-ical traitsrdquo DNA Research vol 16 no 6 pp 311ndash323 2009

[16] L Li and X Y Zheng ldquoThe development of multiplex EST-SSRmarkers to identification Chinese cabbage [Brassica campestrisL chinensis (L) Makino and Brassica campestris L pekinensis(Lour) Olsson] cultivarsrdquo Acta Horticulturae Sinica vol 37 no11 pp 1627ndash1634 2009

[17] F L Iniguez-Luy L Lukens M W Farnham R M Amasinoand T C Osborn ldquoDevelopment of public immortal mappingpopulations molecular markers and linkage maps for rapidcycling Brassica rapa and B oleraceardquo Theoretical and AppliedGenetics vol 120 no 1 pp 31ndash43 2009

[18] H Feng P Wei Z-Y Piao et al ldquoSSR and SCAR mapping ofa multiple-allele male-sterile gene in Chinese cabbage (Brassicarapa L)rdquo Theoretical and Applied Genetics vol 119 no 2 pp333ndash339 2009

[19] X Li N Ramchiary S R Choi et al ldquoDevelopment ofa high density integrated reference genetic linkage map forthe multinational Brassica rapa Genome Sequencing ProjectrdquoGenome vol 53 no 11 pp 939ndash947 2010

[20] S K Parida D K Yadava and T Mohapatra ldquoMicrosatellitesin Brassica unigenes relative abundance marker design anduse in comparative physical mapping and genome analysisrdquoGenome vol 53 no 1 pp 55ndash67 2010

[21] Z Wang J Li Z Luo et al ldquoCharacterization and developmentof EST-derived SSRmarkers in cultivated sweetpotato (Ipomoeabatatas)rdquo BMC Plant Biology vol 11 article 139 2011

[22] W Li J Zhang Y Mou et al ldquoIntegration of Solexa sequenceson an ultradense genetic map in Brassica rapa Lrdquo BMCGenomics vol 12 article 249 2011

[23] J Zou D Fu H Gong et al ldquoDe novo genetic variation associ-ated with retrotransposon activation genomic rearrangementsand trait variation in a recombinant inbred line population ofBrassica napus derived from interspecific hybridization withBrassica rapardquo Plant Journal vol 68 no 2 pp 212ndash214 2011

[24] H Bagheri M El-Soda I van Oorschot et al ldquoGenetic analysisofmorphological traits in a new versatile rapid-cyclingBrassicarapa recombinant inbred line populationrdquo Frontiers in PlantScience vol 3 article 183 2012

[25] J Shi S Huang J Zhan et al ldquoGenome-wide microsatellitecharacterization and marker development in the sequencedBrassica crop speciesrdquo DNA Research vol 21 no 1 pp 53ndash682014

[26] W Powell M Morgante C Andre et al ldquoThe comparisonof RFLP RAPD AFLP and SSR (microsatellite) markers forgermplasm analysisrdquoMolecular Breeding vol 2 no 3 pp 225ndash238 1996

[27] R K Varshney A Graner and M E Sorrells ldquoGenicmicrosatellite markers in plants features and applicationsrdquoTrends in Biotechnology vol 23 no 1 pp 48ndash55 2005

[28] I EujaylMK Sledge LWang et al ldquoMedicago truncatulaEST-SSRs reveal cross-species genetic markers for Medicago spprdquoTheoretical and Applied Genetics vol 108 no 3 pp 414ndash4222004

[29] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005

[30] M C Saha J D Cooper M A R Mian K Chekhovskiy andG DMay ldquoTall fescue genomic SSRmarkers development andtransferability across multiple grass speciesrdquo Theoretical andApplied Genetics vol 113 no 8 pp 1449ndash1458 2006

[31] R K Varshney R Sigmund A Borner et al ldquoInterspecifictransferability and comparative mapping of barley EST-SSRmarkers in wheat rye and ricerdquo Plant Science vol 168 no 1pp 195ndash202 2005

[32] J E Zalapa H Cuevas H Zhu et al ldquoUsing next-generationsequencing approaches to isolate simple sequence repeat (SSR)loci in the plant sciencesrdquoThe American Journal of Botany vol99 no 2 pp 193ndash208 2012

[33] L Li W M He L P Ma et al ldquoConstruction Chinesecabbage (Brassica rapa L) core collection and its EST-SSRfingerprint database by EST-SSRmolecular markersrdquoGenomicsand Applied Biology vol 28 pp 76ndash88 2009


[34] Y Xin H Cui M Lu et al ldquoData mining for SSRs in ESTsand EST-SSR marker development in Chinese cabbagerdquo ActaHorticulturae Sinica vol 33 no 3 pp 549ndash554 2006

[35] Y Ge N Ramchiary T Wang et al ldquoDevelopment and linkagemapping of unigene-derived microsatellite markers in Brassicarapa Lrdquo Breeding Science vol 61 no 2 pp 160ndash167 2011

[36] N Ramchiary V D Nguyen X Li et al ldquoGenic microsatel-lite markers in brassica rapa development characterizationmapping and their utility in other cultivated and wild brassicarelativesrdquo DNA Research vol 18 no 5 pp 305ndash320 2011

[37] F Wang L Li H Li et al ldquoTranscriptome analysis of rosetteand folding leaves in Chinese cabbage using high-throughputRNA sequencingrdquo Genomics vol 99 no 5 pp 299ndash307 2012

[38] S Wang X Wang Q He et al ldquoTranscriptome analysis of theroots at early and late seedling stages using Illumina paired-endsequencing and development of EST-SSR markers in radishrdquoPlant Cell Reports vol 31 no 8 pp 1437ndash1447 2012

[39] G Pertea X Huang F Liang et al ldquoTIGR gene indicesclustering tools (TGICL) a software system for fast clusteringof large EST datasetsrdquo Bioinformatics vol 19 no 5 pp 651ndash6522003

[40] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 p 407 1993

[41] M G Grabherr B J Haas M Yassour et al ldquoFull-lengthtranscriptome assembly fromRNA-Seq datawithout a referencegenomerdquoNature Biotechnology vol 29 no 7 pp 644ndash652 2011

[42] Z Wang B P Fang J Chen et al ldquoDe novo assembly and char-acterization of root transcriptome using Illumina paired-endsequencing and development of cSSR markers in sweetpotato(Ipomoea batatas)rdquo BMC Genomics vol 11 article 726 2010

[43] W Wei X Qi L Wang et al ldquoCharacterization of the sesame(Sesamum indicum L) global transcriptome using Illuminapaired-end sequencing and development of EST-SSR markersrdquoBMC Genomics vol 12 article 451 2011

[44] X Liang X Chen Y Hong et al ldquoUtility of EST-derived SSRin cultivated peanut (Arachis hypogaea L) and Arachis wildspeciesrdquo BMC Plant Biology vol 9 article 35 2009

[45] S Dutta G Kumawat B P Singh et al ldquoDevelopment of genic-SSR markers by deep transcriptome sequencing in pigeonpea[Cajanus cajan (L) Millspaugh]rdquo BMC Plant Biology vol 11article 17 2011

[46] K D Scott P Eggler G Seaton et al ldquoAnalysis of SSRs derivedfrom grape ESTsrdquoTheoretical and Applied Genetics vol 100 no5 pp 723ndash726 2000

[47] K Shirasawa K Ishii C Kim et al ldquoDevelopment of CapsicumEST-SSR markers for species identification and in silico map-ping onto the tomato genome sequencerdquo Molecular Breedingvol 31 no 1 pp 101ndash110 2013

[48] S Cloutier Z Niu R Datla and S Duguid ldquoDevelopmentand analysis of EST-SSRs for flax (Linum usitatissimum L)rdquoTheoretical and Applied Genetics vol 119 no 1 pp 53ndash63 2009

[49] R K Aggarwal P S Hendre R K Varshney P R Bhat VKrishnakumar and L Singh ldquoIdentification characterizationand utilization of EST-derived genic microsatellite markers forgenome analyses of coffee and related speciesrdquo Theoretical andApplied Genetics vol 114 no 2 pp 359ndash372 2007

[50] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 article 278 2010

[51] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005

[52] K N Yamini K Ramesh V Naresh P Rajendrakumar KAnjani andV Dinesh Kumar ldquoDevelopment of EST-SSRmark-ers and their utility in revealing cryptic diversity in safflower(Carthamus tinctorius L)rdquo Journal of Plant Biochemistry andBiotechnology vol 22 no 1 pp 90ndash102 2013

[53] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003

[54] M Wen H Wang Z Xia M Zou C Lu and W WangldquoDevelopmenrt of EST-SSR and genomic-SSRmarkers to assessgenetic diversity in Jatropha Curcas Lrdquo BMC Research Notesvol 3 article 42 2010

[55] S Tang RAOkashahM-MCordonnier-Pratt et al ldquoEST andEST-SSR marker resources for Irisrdquo BMC Plant Biology vol 9article 72 2009

[56] J Blanca J Canizares C Roig P Ziarsolo F Nuez and BPico ldquoTranscriptome characterization and high throughputSSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae)rdquoBMC Genomics vol 12 article 104 2011

[57] T Liu S Zhu L Fu et al ldquoDevelopment and characterizationof 1827 expressed sequence tag-derived simple sequence repeatmarkers for ramie (Boehmeria nivea L Gaud)rdquo PLoS ONE vol8 no 4 Article ID e60346 pp 1091ndash1104 2013

[58] H Pan J Wang Y Wang Z Qi and S Li ldquoDevelopment andmapping of EST-SSR markers in wheatrdquo Scientia AgriculturaSinica vol 43 pp 452ndash461 2010

[59] H Sonah R K Deshmukh A Sharma et al ldquoGenome-widedistribution and organization of Microsatellites in plants aninsight into marker development in Brachypodiumrdquo PLoS ONEvol 6 no 6 Article ID e21298 2011

[60] J H Peng and N L V Lapitan ldquoCharacterization of EST-derived microsatellites in the wheat genome and developmentof eSSR markersrdquo Functional and Integrative Genomics vol 5no 2 pp 80ndash96 2005

[61] C Wang W Guo C Cai and T Zhang ldquoCharacterizationdevelopment and exploitation of EST-derived microsatellites inGossypium raimondii Ulbrichrdquo Chinese Science Bulletin vol 51no 5 pp 557ndash561 2006

[62] R Singh N M Zaki N-C Ting et al ldquoExploiting an oil palmEST database for the development of gene-derived SSRmarkersand their exploitation for assessment of genetic diversityrdquoBiologia vol 63 no 2 pp 227ndash235 2008

[63] A Stagel E Portis L Toppino G L Rotino and S LanterildquoGene-based microsatellite development for mapping and phy-logeny studies in eggplantrdquo BMC Genomics vol 9 article 3572008

[64] L Cardle L Ramsay D Milbourne M Macaulay D Marshalland RWaugh ldquoComputational and experimental characteriza-tion of physically clustered simple sequence repeats in plantsrdquoGenetics vol 156 no 2 pp 847ndash854 2000

[65] L Gao J Tang H Li and J Jia ldquoAnalysis of microsatellitesin major crops assessed by computational and experimentalapproachesrdquo Molecular Breeding vol 12 no 3 pp 245ndash2612003

[66] R K Varshney T Thiel N S P Langridge and A Graner ldquoInsilico analysis on frequency and distribution of microsatellites


in ESTs of some cereal speciesrdquo Cellular and Molecular BiologyLetters vol 7 no 2 pp 537ndash546 2002

[67] M La Rota R V Kantety J-K Yu and M E SorrellsldquoNonrandomdistribution and frequencies of genomic and EST-derived microsatellite markers in rice wheat and barleyrdquo BMCGenomics vol 6 article 23 2005

[68] M Morgante M Hanafey and W Powell ldquoMicrosatellitesare preferentially associated with nonrepetitive DNA in plantgenomesrdquo Nature Genetics vol 30 no 2 pp 194ndash200 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Simple sequence repeat (SSR) markers or microsatel-lite markers are among the most important markers inplants SSRs have been widely used in genetic mappingand molecular breeding in plants because they are highlyabundant and have significant polymorphism Other factorslike accessibility for detection reliability and codominancealso make them perfect markers for such purposes [26]SSRs found in transcribed sequences are called expressedsequence-simple sequence repeats (EST-SSRs) Comparedwith genomic-SSRs detected in noncoding sequences EST-SSRs are more efficient for QTL mapping gene targetingand MAS [27] As transcribed sequences are more conservedthan noncoding sequences the transferability of EST-SSRsis better than genomic-SSRs [28ndash30] which can be utilizedfor cross genome comparison and evolutionary analysis [2731] Additionally abundant ESTs were generated in recentyears with the development of next-generation sequencingapproaches making identification of EST-SSRs more practi-cal and cost-efficient [32] Many EST-SSRs have been iden-tified in Chinese cabbage [16 20 25 33ndash36] Because wholegenome sequencing of Chinese cabbage is still underway newEST-SSRs could also be identified for further studies suchas high-density genetic linkage map construction geneQTLmapping and cultivar identification

In our previous study the whole transcriptomes wereanalyzed for the rosette leaves and folding leaves of atypical heading Chinese cabbage namely FuShanBaoTouusing a SolexaIllumina RNA-Seq platform and a large-scaleEST database was generated [37] In this study we furtherassembled those ESTs from the RL and FL libraries intononredundant unigenes A total of 10420 EST-SSRs wereidentified among which 2744 EST-SSRs are detected forthe first time according to the SSR marker database forBrassica (httpoilcropsinfoSSRdb) We characterized theseidentified EST-SSRs and designed 7877 PCR primer pairs for1561 EST-SSRs Furthermore serving as a validation purposewe tested polymorphisms of 24 EST-SSRs We expect thisstudy can pave the road for further investigation of new EST-SSR markers and for construction of high-density geneticmaps

2 Materials and Methods

21 Plant Materials For EST-SSR identification and primerdesign a typical heading Chinese cabbage namely FuShan-BaoTou was used in this study For primer assessmentand SSR polymorphism analysis a panel of twenty-fourcultivars of Chinese cabbage was used including nineteenmorphologically diverse cultivars of Brassica rapa L ssppekinensis (B pekinensis L) and five Brassica rapa L chinensis(B chinensis L) All plants were grown in a greenhouse with168 photoperiod at 22plusmn2∘C Leaves were collected after theywere grown for two weeks from ten seedlings of each cultivarand were pooled together for DNA extraction

22 De Novo Assembly We assembled the clean read datasetpresented by Wang et al [37] from the RL and FL librariesaccording to the methods described byWang et al [38] usingthe Trinity software (httptrinityrnaseqsourceforgenet)

Contigs and unigenes were obtained from these two librariesrespectively Redundant sequences were removed and over-lapping unigenes were assembled into continuous sequencesby the TIGR Gene Indices Clustering (TGICL) tools [39]Similarity was set at 94 and an overlap length was set at100 bp

23 Identification of EST-Derived SSRs and Primer DesignSSRs were detected with the MicroSAtellite software (MISAhttppgrcipk-gaterslebendemisa) Parameters were setwith a minimum number of 12 6 5 5 4 and 4 repeatunits for identification of mono- di- tri- tetra- penta- andhexanucleotide motifs respectively Primers were designedusing primer 3 with no SSR allowed in primers Primer lengthranged from 18 to 28 bp (with an optimality at 23) Annealingtemperature was set at 55ndash65∘C (with an optimality at 60∘C)The size of a PCR product ranged from 80 to 300 bp

24 Mapping EST-SSRs The physical positions of theEST-SSRs identified in the study were determined byaligning the SSRs and flanking sequences (50 bp at eachside) to the Brassica rapa (Chiifu-401) reference genome(httpbrassicadborgbrad) using BLASTN New EST-SSRs were identified by comparing with previouslyreported SSRs in the SSR marker database for Brassica(httpoilcropsinfoSSRdb) [25]

25 SSRAmplification and SSR PolymorphismAnalysis DNAwas extracted following a CTAB DNA extraction protocol[40] The DNA sample of the Chinese cabbage FuShan-BaoTou was used as template to detect the availability ofSSR primers designed above The DNA samples of thoseaforementioned twenty-four cultivars of Chinese cabbagewere used as templates for SSR polymorphism analysis Thepolymorphisms of EST-SSRs were validated by 6 denatur-ing polyacrylamide gel electrophoresis 12 nondenaturingpolyacrylamide gel electrophoresis and sequencing

3 Results

31 De Novo Assembly High quality clean read data fromthe RL and FL libraries by Wang et al [37] were assembledusing the Trinity software package [41] A total of 99684 and95411 contigs were obtained with an average length of 333and 342 bp and amedian length (N50) of 531 and 536 bp fromthe RL and FL libraries respectively (Table 1)

Contigs from the same transcript were detected withpaired-end reads as well as the distances between thesecontigs Using the Trinity software package we assembledthese contigs into unigenes in whichNswere removedTheseunigenes were set to be not extendable on either end of thesequences A total of 46294 and 48473 unigenes from theRL and FL libraries were obtained with an average lengthof 707 and 680 bp and a median length (N50) of 1000 and980 bp respectively (Table 1) Size distribution of the contigsand unigenes is consistent with the RL and FL libraries asshown in Figure 1 indicating that our Illumina sequencingsolution is reliable and reproducible Unigenes from the twosamples were combined redundant unigenes were removed






UnigeneRL 46294 32729586 707 1000 46294 19512 26782FL 48473 32971187 680 980 48473 19749 28724All 51694 40724256 788 1154 51694 23850 27844

RLFL

1

10

100

1000

10000

RLFL

All unigenes

1

10

100

1000

10000

100000

100000

Contig

Unigene

Num

ber o

f rea

ds

Num

ber o

f rea

ds

Sequence size (bp)

Sequence size (bp)

le200

201

ndash300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

140

1ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000

le300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

1401

ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000









1644

4043

4405

90 112 1260

1000

2000

3000

4000

5000

Num

ber o

f EST

-SSR

s

Mon

onuc

leot

ide

Din

ucle

otid

e

Trin

ucle

otid

e

Tetr

anuc

leot

ide

Pent

anuc

leot

ide

Hex

anuc

leot

ide












150

08 33

338

17 44

139

08 38

08 08 30

74 60

13 31

00

50

100

150

200

250

300

350

400

Freq

uenc

y (

)

Motif

AT

CG

ACG

T

AGC

T

ATA

T

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

T

ACG

CG

T

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

G

Oth

ers





(Table s6)



355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR




4 Discussion







BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






UnigeneRL 46294 32729586 707 1000 46294 19512 26782FL 48473 32971187 680 980 48473 19749 28724All 51694 40724256 788 1154 51694 23850 27844

RLFL

1

10

100

1000

10000

RLFL

All unigenes

1

10

100

1000

10000

100000

100000

Contig

Unigene

Num

ber o

f rea

ds

Num

ber o

f rea

ds

Sequence size (bp)

Sequence size (bp)

le200

201

ndash300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

140

1ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000

le300

301

ndash400

401

ndash500

501

ndash600

601

ndash700

701

ndash800

801

ndash900

901

ndash1000

1001

ndash1100

1101

ndash1200

1201

ndash1300

1301

ndash1400

1401

ndash1500

1501

ndash1600

1601

ndash1700

1701

ndash1800

1801

ndash1900

1901

ndash2000

2001

ndash2100

2101

ndash2200

2201

ndash2300

2301

ndash2400

2401

ndash2500

2501

ndash2600

2601

ndash2700

2701

ndash2800

2801

ndash2900

2901

ndash3000

ge3000









1644

4043

4405

90 112 1260

1000

2000

3000

4000

5000

Num

ber o

f EST

-SSR

s

Mon

onuc

leot

ide

Din

ucle

otid

e

Trin

ucle

otid

e

Tetr

anuc

leot

ide

Pent

anuc

leot

ide

Hex

anuc

leot

ide












150

08 33

338

17 44

139

08 38

08 08 30

74 60

13 31

00

50

100

150

200

250

300

350

400

Freq

uenc

y (

)

Motif

AT

CG

ACG

T

AGC

T

ATA

T

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

T

ACG

CG

T

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

G

Oth

ers





(Table s6)



355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR




4 Discussion







BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




1644

4043

4405

90 112 1260

1000

2000

3000

4000

5000

Num

ber o

f EST

-SSR

s

Mon

onuc

leot

ide

Din

ucle

otid

e

Trin

ucle

otid

e

Tetr

anuc

leot

ide

Pent

anuc

leot

ide

Hex

anuc

leot

ide












150

08 33

338

17 44

139

08 38

08 08 30

74 60

13 31

00

50

100

150

200

250

300

350

400

Freq

uenc

y (

)

Motif

AT

CG

ACG

T

AGC

T

ATA

T

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

T

ACG

CG

T

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

G

Oth

ers





(Table s6)



355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR




4 Discussion







BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




150

08 33

338

17 44

139

08 38

08 08 30

74 60

13 31

00

50

100

150

200

250

300

350

400

Freq

uenc

y (

)

Motif

AT

CG

ACG

T

AGC

T

ATA

T

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

T

ACG

CG

T

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

G

Oth

ers





(Table s6)



355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR




4 Discussion







BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


355

444

133

69

0

10

20

30

40

50

CDS Uncertain

Freq

uenc

y (

)

5998400UTR 3

998400UTR




4 Discussion







BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


BR-es3

145bp

160bp163bp

99bp

BR-es6 130bp160bp

BR-es7148bp

BR-es12

144bp140bp

135bp

117 bp


101 bpBR-es14

130bp125 bp

120 bp

128 bp

BR-es19

151bp

147bp

BR-es16

191bp187 bp

169bp

193bp

185 bp

189 bp



134bp

BR-es17

141bp

123 bp

117 bp

BR-es18 155bp131bp

143bp

161bp

137bp149bp

BR-es4 127 bp

146bp

BR-es5 167bp

161bp

151bp

BR-es9 182 bp 155bp


133bp

115 bp

BR-es8

141bp

129 bp122 bp

BR-es1 97bp91bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24















Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Acknowledgments


References
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Characterization and Development of EST-SSRs …downloads.hindawi.com/journals/ijg/2015/473028.pdf · 2019-07-31 · Research Article Characterization and Development