research article construction and characterization of a...

Research ArticleConstruction and Characterization of a Bacterial ArtificialChromosome Library for the Hexaploid Wheat Line 92R137

Qingdong Zeng1 Fengping Yuan2 Xin Xu2 Xue Shi2 Xiaojun Nie2

Hua Zhuang1 Xianming Chen3 Zhonghua Wang2 Xiaojie Wang1 Lili Huang1

Dejun Han2 and Zhensheng Kang1

1 State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection Northwest AampF University YanglingShaanxi 712100 China

2 State Key Laboratory of Crop Stress Biology for Arid Areas and College of Agronomy Northwest AampF University YanglingShaanxi 712100 China

3Wheat Genetics Quality Physiology and Disease Research Unit Agricultural Research ServiceUnited States Department of Agriculture and Department of Plant Pathology Washington State University PullmanWA 99164-6430 USA

Correspondence should be addressed to Zhensheng Kang kangzsnwsuafeducn

Received 28 January 2014 Revised 26 March 2014 Accepted 18 April 2014 Published 5 May 2014

Academic Editor Guihua H Bai

Copyright copy 2014 Qingdong Zeng et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137 a bacterial artificial chromosome(BAC) library including two sublibraries was constructed using the genomic DNA of 92R137 digested with restriction enzymesHindIII and BamHI The BAC library was composed of total 765696 clones of which 390144 were from the HindIII digestionand 375552 from the BamHI digestion Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selectedfrom the HindIII sublibrary and 573 clones from the BamHI sublibrary the average insert sizes were estimated as 129 and 113 kbrespectivelyThus theHindIII sublibrary was estimated to have a 301-fold coverage and the BamHI sublibrary a 253-fold coveragebased on the estimated hexaploid wheat genome size of 16700Mb The 765696 clones were arrayed in 1994 384-well plates Allclones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools The probability of identifying aclone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated tobe more than 996 Through polymerase chain reaction screening the 5D pools with Xwe173 a marker tightly linked to Yr26 sixBAC clones were successfully obtainedThese results demonstrate that the BAC library is a valuable genomic resource for positionalcloning of Yr26 and other genes of interest

1 Introduction

Providing about 20 of calories consumed by humans wheat(Triticum aestivum L) feeds 35 of the worldrsquos population asone primary food [1 2] Wheat stripe (yellow) rust causedby Puccinia striiformis Westend f sp tritici Erikss (Pst) isone of the most destructive diseases worldwide [3 4] Chinais considered to be the largest individual region sufferingstripe rust damage in the world and its annual wheat yieldloss caused by the disease is about one million metric tons

on average [5] Growing resistant cultivars is considered tobe the most economically effective and environmentally safemeasure for control of stripe rust [6 7] For developingresistant cultivars wheat germplasm with effective resistanceand genes conferring the resistance should be identified

92R137 is a T aestivum and Haynaldia villosa 6VS6ALtranslocation line of hexaploid wheat developed by theCytogenetics Institute of Nanjing Agricultural University [8]The line possesses a cloned gene Pm21 on the translocatedchromosomal segment 6VS for resistance to powderymildew

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 845806 9 pageshttpdxdoiorg1011552014845806

2 BioMed Research International

caused by Blumeria graminis f sp tritici (Bgt) [9] It has astripe rust resistance gene Yr26 which is effective againstpredominant races of Pst in China [8 10ndash13] Although Yr26is identified as susceptible to a new virulent pathotype it canprovide good protection of crops from the Pst populationwhen combined with other resistance genes because thevirulent pathotype has a narrow spectrum of virulence[14] Because 92R137 has resistance to both stripe rust andpowdery mildew the two most important wheat diseasesin China the line has been widely used in wheat breedingprograms in China [8] Cloning Yr26 can contribute tothe understanding of molecular mechanisms of stripe rustresistance and provide basis for wisely using the gene andother genes to achieve long lasting and high level resistancesto stripe rust

A high-resolution map has been developed for Yr26 ina population developed with 92R137 [14] Among a largenumber of markers linked with Yr26 codominant markerXwe173 which was developed from wheat EST BF474347 [8]was found to be only 14 cM away and appeared to be reliablefor marker-assisted detection of the gene [15] Because Yr26is mapped close to the centromere of chromosome 1B [8] it isdifficult to identify closer markers Although we are currentlyusing a much larger population (composed of nearly 10000F2individuals) for identifying closer markers a bacterial

artificial chromosome library (BAC) developed from theresistant wheat line should speed up the process of cloningYr26

In Triticeae seven genes for resistance to rusts are clonedincluding Lr1 [16] Lr10 [17] and Lr21 [18] for resistance toleaf rust Sr33 [19] and Sr35 [20] for resistance to stem rustYr36 [1] for resistance to stripe rust and Lr34Yr18 conferringresistance to both leaf rust and stripe rust [21] These molec-ularly very diverse genes were cloned all using themap-basedcloning approach Therefore we have taken the approach forcloning Yr26 in 92R137 This approach requires establishingphysicalmaps using a large insert library Among the differentkinds of libraries a BAC library is a preferred system overother large insert libraries for its advantage of easy operationhigh stability and low chimerism [22]

Although several protocols of BAC library construction[22ndash28] and BAC libraries for tetraploid wheat and hexaploidwheat [29ndash35] have been published research progress fordevelopment of large insertion libraries for a large genomespecies is still limited Particularly hexaploidwheat has a verylarge genome of 167 Gb [36] In various reports differentmethods were adopted to improve transformation efficiencyand quality such as multiple selection to remove small DNAmolecules [37] electroelution means to recover size-selectedDNA from agarose gel [24] and using TAE instead of TBEbuffer in electroelution [26 38] In the present study we triedto integrate almost all these modified methods to construct ahigh quality BAC library for 92R137 aimed at cloningYr26 Toimprove genome representation and reduce the gaps betweenBAC contigs resulting from uneven distribution of restrictionsites [39]HindIII and BamHI were used separately to obtainBAC clones To speed up screening the BAC for 92R137 a new5D clone pooling scheme [40] was adopted and proven to bemore efficient

2 Materials and Methods

21 Plant Material and HMW DNA Preparation Springwheat 92R137 kindly provided by Professor Peidu Chen attheNanjingAgricultural University was used for BAC libraryconstruction Seeds were sown in pots filled with soil mixtureand plants were grown in dark at room temperature Whenthey grewup to 10 cm long seedlingswere harvested forDNAextraction

Twenty-five gram leaf tissue was ground into fine powderin liquid nitrogen following the slightly modified proceduredescribed by Luo and Wing [28] to release their intactnuclei The nuclei were isolated and purified four times withice-cold NIBM (10mM Tris-HCl with a pH of 80 10mMEDTA with a pH of 80 100mM KCl 05M sucrose 4mMspermidine 1mM spermine with 10 Triton X-100 and01 120573-mercaptoethanol) at 10mL1 g tissue and centrifugedat a force set to 1800 g The obtained pellet was gentlyresuspended in approx 1mL NIB (10mM Tris-HCl pH 8010mM EDTA pH of 80 100mM KCl 05M sucrose 4mMspermidine and 1mM spermine with 10 Triton X-100)At the equal volume 1 (wv) low melting temperature(LMP) agarose (Amresco Solon OH USA) dissolved in NIBwas added into the suspension and the mixture was pouredinto a 100120583L Plug Mold (Bio-Rad Hercules CA USA)After adding lysis buffer 05molL EDTA with a pH of 901 (wv) N-lauroylsarcosine and proteinase K at 1mgmL(Amresco) the plugs were rotated in an incubator at 50∘Cfor about 48 h and at 24 h the buffer was changed with freshbuffer Then the agarose plugs were rinsed twice with ice-cold T

10E10

buffer (10mmolL Tris-HCl with a pH of 8010mmolL EDTA with a pH of 80) containing 1mmolLphenylmethylsulphonyl fluoride (Sigma St Louis MO USA)and twice with ice-cold TE buffer 1 hour each rinse Thenthe plugs were stored in TE buffer (pH 80) at 4∘C for futureuse

22 Restriction Digestion and DNA Fragment Size SelectionTo determine the optimum conditions for the highest per-centage of fragments between 120 and 2425 kb a series ofeight partial digests were performed One half of the DNAplug was chopped into fine pieces as one treatment andequilibrated with 45120583L of restriction enzyme buffer for 30minutes 5 120583L enzyme diluents with 0 04 08 12 16 20 24and 10U BamHI or HindIII (New England Biolabs IpswichMA USA) were added into the treatment contained inmicrocentrifuge tubes and placed on ice for 30minutesThenthe microcentrifuge tubes were incubated in 37∘C water bathfor 30 minutes for digestion After adding 10 120583L 05molLEDTA (pH 80) the tubes were incubated on ice for at least10min to stop digestion

The plug pieces were separated in a 1 agarose gel in 05xTBE using aCHEFDR-III System (Bio-Rad) at 6Vcm 1ndash50 sswitch times linear ramp 120∘ angle at 14∘C for 18 h Once theoptimal digestion condition for one batch of plugs to producefragments between 120 and 2425 kbwas identified the partialdigestions of 6 plugs were performed under the optimaldigestion condition DNA fragments with sizes ranging from120 to 2425 kb were excised (corresponding to 120ndash180 and

BioMed Research International 3

180ndash2425 kb measured by a ruler) and reloaded onto new 1agarose gel in 05x TBE and run at 6Vcm 3ndash5 s switch timeslinear ramp 120∘ angle at 14∘C for 18 h to select them by sizeonce again [27] After the agarose slices containing fragmentslarger than 120 kb were recovered the DNAwas electroelutedfor 35 h at 45 Vcm in electrophoresis cell (JY-SP-C JUNYIBeijing) The polarities of electrophoresis cell were reversedat 4∘C for 1min [24] The concentrations of the electroelutedDNA were measured by agarose gel electrophoresis with the120582 DNAmarker

23 Vectors The library construction vectors were 81 kbCopyControl pCC1BAC HindIII Cloning-Ready Vectors and81 kb CopyControl pCC1BAC BamHI Cloning-Ready Vec-tors (Epicentre Madison WI USA) Both vectors allowfor the LacZ based blue-white selections of recombinantclones and the selection of chloramphenicol resistance as anantibiotic selectable markers

24 Ligation and Bacterial Transformation The ligationswere individually carried out in a 100 120583L reaction solutionaccording to themanufacturerrsquos instructions First 25 ng vec-tors about 100 ng electroeluted HMWDNA and appropriateddH2O were mixed to form an 84 120583L solution and incubated

at 65∘C for 10minThe solution was cooled to 4∘C and addedwith 10 120583L 10x Fast-Link ligation buffer 1 120583L 100mM ATPand 2 120583L Fast-Link DNA ligase The ligation solutions wereincubated at 16∘C for 16 h Finally the reaction solutionswere heated to 65∘C for 15min to inactivate Fast-Link DNAligase and desalted with an agarose cone on ice for 15 h[41]

The transformation of the ligation product into Trans-forMax EPI300 Electrocompetent E coli cells (Epicentre)was carried out in 01 cm cuvettes by electroporation usinga MicroPulser Electroporation Apparatus (Bio-Rad) at a16 kV (119864 = 16 kVcm) [38] One microliter of the lig-ation product and 20120583L of the transformed EPI300 cellswere gently blended in a wide-bore pipette tip for elec-troporation Immediately the cells were transferred into1mL SOC medium [28] and incubated at 37∘C for 1 hwhile shaken at 200 rpm [22] The cells were placed on LBmedium containing 125 120583gmL chloramphenicol 90120583gmLX-Gal and 90 120583gmL IPTG [27] and incubated at 37∘C for20 h

25 Isolation and Determination of Insert DNA The recom-binant clone number per microliter of the ligation productwas determined by counting white colonies If the numberwas above 500 and blue colonies were less than 20 [27] 20ndash24 white colonies were randomly sampled from each ligationand allowed to grow in 4mL LB containing 125120583gmLchloramphenicol 37∘C for 16 h while shaken at 200 rpmPlasmid DNA isolation was conducted following a modifiedalkaline lysis protocol [27] The plasmid DNA was digestedwith 5U of NotI (Fermentas) at 37∘C for 4 h and loaded onto1 agarose in 05x TBE CHEF gel (6Vcm 5ndash15 s pulse timefor 16 h at 14∘C) to determine the insert size 120582 PFG Marker

(New England Biolabs) along with the Quantity One (Bio-Rad V462) was used for molecular weight determination

26 Picking and Storing BAC Clones If the test coloniesmet the requirements in average insert size (gt100 kb) andempty vector rate (lt5) all ligated DNAs were transformedinto EPI300 competent cells Individual white colonies weremanually pickedwith sterilized toothpicks andplaced inwellsof 384-well plates containing freezing media [28] The platescarrying white colonies were incubated at 37∘C for 20 h anda duplicate copy of the primary BAC library was made byinoculation of clones from the primary source plates to newplates with a hand-holding replicator and the new plateswere cultured under the same conditions The primary andsecondary copies were stored at minus80∘C

27 Testing Chloroplast Contamination To test the chloro-plast contamination of the BAC library a set of PCRprimers was designed for three selected chloroplast genesThe first gene encodes a NADH dehydrogenase and theprimer pair of 5ndh (31015840-GGGTAGAGGTAGAAACTATC-51015840) and 3ndh (31015840-CGCTTCTGAATTGATCTCATCC-51015840) wasused to amplify a 14 kb fragment The second geneencodes a Photosystem II protein and the primer pair of5psb (31015840-GGAAGCTGCATCTGTTGATG-51015840) and 3psb (31015840-AGGGAAGTTGTGAGCATTACG-51015840) was used to amplifyan 800 bp fragmentThe third gene encodes the Ribulose-15-bisphosphate carboxylase large subunit and the primer pair5rbcL (31015840-CTGATACTTGGCAGCATTCC-51015840) and 3rbcL(31015840-CGATTAGCTGCTGCACCAG-51015840) was used to amplify a12 kb fragment [22] The genomic DNA of 92R137 was usedas the positive control

28 Pooling BAC Clones The improved 5D clone poolingstrategy [40] was used to make pools of BAC clones The5D pools contained pools of the 3D traditional grid designrow-pools (RP) column-pools (CP) and plate-pools (PP)and 2D super pools the row super-pools (RSP) and columnsuper-pools (CSP) of the plate-pools To obtain a plate-poolBAC clones in the wells of each 384-well plate were dippedinto 30mL LB broth containing 125mgL chloramphenicolwith the 384-pin replicator to inoculate the clones on aLB broth After growing overnight the clone cells wereprecipitated by centrifugation and resuspended with 1mL10 glycerol and stored at minus80∘C for future use In sucha way a total of 1994 plate pools were produced PlasmidDNA was isolated from 100 120583L cell culture of each poolfollowing the standard alkaline lysis protocol The plate-poolDNA was dissolved in 1x TE buffer and the concentrationwas measured with a Nanodrop 2000 spectrophotometer(Nano-drop Technologies Wilmington DE) at a wavelengthof 260 nm

To construct super pools the 1994 plate pools weredivided into 20 primary BAC groups each of which contained100 BAC plates (arranged in a grid consisting of 10 rows and10 columns)


Lane

485

97

1455

1942425

2913395

388

(kb)

HindIII unitshalf plug 0 2 4 6 8 10 12 501 2 3 4 5 6 7 8 M

(a)

485

97

1455

19424252913395388

(kb)

BamHI unitshalf plug0 2 4 6 8 10 12 501 2 3 4 5 6 7 8 M Lane

(b)

Figure 1 Partial digestion of DNA in half plugs Lanes 1ndash8 contain DNA samples digested with restriction enzyme at the increasingly higherconcentrations (a) Partial digestions of half DNA plugs with serial dilutions ofHindIII at 37∘C for 30min (b) Partial digestions of half DNAplugs with serial dilutions of BamHI at 37∘C for 30min Plug pieces were separated on 1 agarose gel in 05x TBE and run in the CHEFDR-IIISystem (Bio-Rad) at 6Vcm 1ndash50 s switch time linear ramp 120∘ angle at 14∘C for 18 h M the 120582 PFG marker

Same amounts of the DNAs of the plate-pools in one rowor column were pooled to produce a row (column) super-poolThe 2D super-pool grid of each primary group consistedof 20 super-pools (each containing 3840 clones) The DNAsof the super-pools were diluted to 5 ng120583L with deionized-distilled water and employed as the PCR screening templates

For each plate 40 clone-pools (16 row-pools and 24column-pools) were generated by blending 1120583L cell culture ineach row (column) with 3mL LB broth containing 125mgLof chloramphenicol After the cell cultures grew overnightDNAs were isolated following the standard alkaline lysisprotocol and dissolved into 1x TE buffer The concentrationsof the DNAs were measured with a Nanodrop 2000 spec-trophotometer and they were diluted to have a concentrationof 5 ng120583L with deionized-distilled water and used as a PCRtemplate for screening

29 Screening BAC Library with Yr26 Linked MarkersXwe173 a STS marker linked with Yr26 derived from wheatEST BF474347 [8] was used in screening the BAC libraryFour hundred super pools of 20 groups were PCR screenedTheprimersXwe173F (31015840-GGGACAAGGGGAGTTGAAGC-51015840) and Xwe173R (31015840-GAGAGTTCCAAGCAGAACAC-51015840)were synthesized by Sangon Biotech Co Ltd (ShanghaiChina) The PCR was performed in 15 120583L reaction mixcontaining 1x PCR buffer (MgCl

2-free Fermentas Canada)

2mM MgCl2(Fermentas Canada) 02mM dNTPs (Roche

German) 075 unit Taq DNA polymerase (FermentasCanada) and 06 120583M each primer The PCR amplificationwas performed in a Bio-Rad S1000 Thermal Cycler with 96-well fast reaction module The amplification cycling profile

was 94∘C for 4min 35 cycles each consisting of 94∘C for30 s 60∘C for 1min 72∘C for 1min and extension at 72∘C for10minThe genomic DNA of 92R137 was used as the positivecontrol After amplification 3 120583L 6x loading dye (FermentasPittsburgh PA USA) was added to the PCR product Then8 120583L of the PCR product and loading buffer mixture for eachsample was loaded onto 2 agarose gel in 1x TAE bufferand electrophoresed at 5Vcm for about 30min The gel wasstained with 05 120583gmL ethidium bromide for 20min andvisualized under UV light using Molecular Imager Gel DocXR+ System (Bio-Rad USA)

After the plates containing positive BAC clones wereidentified 40 clone-pools (16 RP and 24 CP) of each selectedplate were further screened to identify individual positiveBAC clones under the same PCR reaction condition as forthe super pools The positive BAC clones were individuallyconfirmed under the same PCR conditions All of the PCRamplifications were repeated three times

3 Results

31 Pilot Partial Digestions and Selection of Large DNA Frag-ments The different enzyme concentrations under the sameincubation timeswere used in the studyTheundigestedDNA(Figures 1(a) and 1(b) Lane 1) appeared to have amean lengthgt600 kb and almost no disintegration indicating that thehigh molecular weight (HMW) DNAs had a good integrityAfter an additional amount of the enzyme was added theDNAs were fully digested (lt50 kb) (Figures 1(a) and 1(b)Lane 8) indicating that the DNAs in the plugs were pure


(kb)

14551942425291

97485

(a)

485

97

1455194

(kb)

(b)

Figure 2 Size selections of the partially digested products of genomic DNA of wheat line 92R137 (a) The first size selection of the partiallydigested products of genomic DNA Plug pieces were separated on 1 agarose gel in 05x TBE buffer and run in a CHEF DR-III System (Bio-Rad) at 6Vcm 1ndash50 s switch time linear ramp 120∘ angle at 14∘C for 18 h (b) The second size selection of the partially digested productsof genomic DNA The first size selected fractions were separated on 1 agarose gel in 05x TBE buffer and run at 6Vcm 3ndash5 s switch timelinear ramp 120∘ angle at 14∘C for 18 h M the 120582 PFG marker

enough to digest Because fragments between 120 kb and2425 kb were desirable the reaction conditions adopted inthe lane which appeared to contain the highest amounts offragments within this size range were used in the large-scalerestriction digestion (Figures 1(a) and 1(b) Lane 3)

Once the reaction condition for producing fragmentsbetween 120 and 2425 kb was determined a mass digestionusing several plugs was performed to produce insert DNAfor the construction of a BAC library After electrophoresisthe first size selection of the partially digested productsof genomic DNA of 92R137 was carried out to recovergel fraction containing 120ndash2425 kb fragments (Figure 2(a))The second size selection was performed using the firstsize-selected fractions cut from the center part of the gelfractions corresponding to 120ndash180 kb fragments and 180ndash2425 kb fragments After the second electrophoresis the gelfractions containing DNA fragments larger than 150 kb werelocated with a ruler and recovered from the gel (Figure 2(b))These gel fractions were immediately used in cloning

32 BAC Library Construction and Characterization Afterelectroelution ligation and transformation two BAC con-structs from the genomic DNA of 92R137 were made withBAC vector pCC1BAC using the genomic DNA digestedseparated with HindIII and BamHI The HindIII constructconsisted of 390144 clones placed in 1016 384-well platesFrom these clones 453 clones were randomly selected andexamined on pulsed-field gels to estimate the insert sizes oftheHindIII clonesTheHindIII clones were shown to have anaverage size of 129 kb and a coverage of 301-fold genome ofthe hexaploid wheat species based on the estimated genomesize of 16700Mb [36] The HindIII sublibrary had 14empty clones (Figures 3 and 4) To estimate the insert sizeof the 375552 BamHI clones arrayed in 978 384-well plates

48597

1455194

2425

(kb)

M

Vector

Figure 3 NotI digests of plasmids from randomly selected whitecolonies The enzyme-digested products of selected plasmids withNotI were loaded onto 1 agarose in 05x TBE CHEF gel 6 Vcm5ndash15 s pulse time for 16 h at 14∘C M the 120582 PFG marker

573 clones were randomly picked and examined on pulsed-field gels The BamHI sublibrary had an average clone sizeof 1126 kb representing 253-fold coverage of the hexaploidwheat genome and contained 34 empty clone (Figures 3and 4) The two sublibraries contained a total of 765696clones in 1994 384-well plates and had an estimated coverageof 554-fold genome of hexaploid wheat All clones wereduplicated in 1994 plates The probability of identifying aclone containing an interested gene (such as Yr26) from thelibrary was estimated to be 996

The percentage of contaminated chloroplasts in thelibrary was estimated by screening 846 clones randomlyselected with three pairs of primers designed for threechloroplast genes [22] Among the 846 clones only 6 werepositive for the tested genes indicating that only 071 of theclones were contaminated by chloroplast genes


02468

10121416

Prop

ortio

n of

diff

eren

t ins

ert

Insert size (kb)

HindIII libraryBamHI library

size c

lone

s (

)

0

0ndash10

10

ndash20

20

ndash30

30

ndash40

40

ndash50

50

ndash60

60

ndash70

70

ndash80

80

ndash90

90

ndash100

100

ndash110

110

ndash120

120

ndash130

130

ndash140

140

ndash150

150

ndash160

160

ndash170

170

ndash180

180

ndash190

190

ndash200

gt200

Figure 4 Insert size distribution of randomly selected BAC clonesincluding 453 clones constructed with HindIII (open bars) and 573clones constructed with BamHI (solid bars)

33 BAC Clone Pooling and PCR Screening with Yr26-LinkedMarker Using the 5D pooling strategy 400 super-pools (200RSPs plus 200 CSPs) were constructed From each superpool plasmid DNA was extracted and adjusted to 5 ng120583Lto be used in the PCR screening for identifying BAC clonescontaining Xwe173 A total of 400 PCR reactions using theDNA templates from the 400 super-pools were performedthree times The genomic DNA of 92R137 was used asthe positive control Two positive amplifications of Xwe173were detected in screening the super-pools and two platesH186 (constructed withHindIII) and B634 (constructed withBamHI) were identified to contain the marker (Figure 5(a))

From 40 pools (16 row pools and 24 column pools)constructed from each of the positive plates two positiveamplifications were detected from both column and row poolof clones in plate H186 Similarly two positive amplificationswere detected from the column and three positive amplifica-tions from the rowpools of clones fromplate B634 Finally sixpositive BAC clones (H186H13 H186E15 B634C10 B634C11B634D10 and B634E10) were identified to contain Xwe173(Figure 5(b)) The results suggest that the BAC library issuitable for identifying clones containYr26 or any other genesin line 92R137

4 Discussion

A large insertion library is the foundation for positionalcloning of an interesting gene line Yr26 without referencegenome sequences such as H villosa the donor of Yr26to hexaploid wheat lines Because the advantage of easyhandling stability and low chimerism the BAC librarytechnique has become relatively popular in modern geneticsresearch compared with other large insert clone techniques[22] To create such genetic resource for map-based cloningof stripe rust resistance geneYr26 in wheat line 92R137 a largeBAC library consisting of 765696 clones was constructed bydigesting genomic DNA of 92R137 with HindIII and BamHIThe different cloning sites were chosen to enhance unbiased

genome representation and minimize the numbers of gapsbetween BAC contigs that result from uneven restriction sitedistributions [39 42 43]

Although several BAC libraries are published [22ndash28] itis still technically required to construct a hexaploid wheatlibrary for interesting genes like Yr26 in a particular wheatgenotype Obtaining high quality DNA fragments of propersizes is a key step for BAC library construction Becausetransformation efficiency and insert size are negatively corre-lated small fragments result in remarkably decreased averageinsert sizes thereby causing chromosomalwalking to becomedifficult [22] Although most DNA molecules lt120 kb areremoved by the first size selection some small DNA frag-ments are still trapped by larger ones [23 27] Many studiesconfirmed that two size selections could effectively eliminatesmall restriction fragments resulting in increased averageinsert sizes [26 32 37 44 45] Therefore we conducted thesecond size selection to eliminate smaller DNA moleculesprior to ligation in the present study and proved the effec-tiveness of the second selection

After size selection two major techniques electroelu-tion and digestion with 120573-agarase are commonly used torecover size-selected DNA from agarose gel Some previousstudies reported that higher transformation efficiencies wereachieved with DNAs isolated by electroelution than with120573-agarase [24 26] In electroelution two electrophoresisbuffers TAE and TBE are used however some researchesshow that borate contained in TBE can inhibit T4 DNAligase activity and thus affect cloning and transformationefficiencies [26 38 46] In the present study we adopted themethod of electroelution with TAE buffer and successfullyconstructed a high quality BAC library

The ratio of clones contaminated by chloroplasts in thelibrary was estimated to be 071 using themethod describedby Farrar and Donnison [22] Compared with those inother BAC libraries for hexaploid wheat this percentagewas slightly higher than those reported by Ratnayaka etal [34] and Shen et al [35] but much lower than 22described by Nilmalgoda et al [32] The low percentage ofchloroplast clones in the library of our study was achievedmainly with young leaves growing in dark thus reducing theirchloroplasts

Integrating genetic and physical maps is a prerequisitefor conducting map-based gene cloning but the integrationneeds the essential step of screening a suitable BAC libraryThe efficiency of BAC library screening can be improvedby adopting an appropriate BAC pooling strategy [47 48]The present study fulfilled this requirement by making poolsof the clones according to the 5D clone pooling strategydescribed by You et al [40] Through 400 PCR reactions765696 clones were efficiently tested with the Yr26-linkedmarker Xwe173 The simple easy and high-throughput PCRscreening methods for identifying BAC clones are reportedin many investigations [22 32 33 37] using different poolingschemes The implementation of the 5D BAC clone poolingstrategy is a highly efficient low cost and rapid approach toscreen a BAC library To validate the selected clones eachof them was confirmed through repeated PCR reactions Toachieve specific amplifications the annealing temperature


92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)

Figure 5 Screening of the BAC library with the Yr26-linked marker Xwe173 (a) Screening of BAC plate pools A 451-bp band was amplifiedin positive control 92R137 withXwe173 Two positive BAC plates H186 (constructed withHindIII) and B634 (constructed with BamHI) wereidentified (b) Six BAC clones (H186H13 H186E15 B634C10 B634C11 B634D10 and B634E10) which were identified from plates H186 andB634 were confirmed to contain Xwe173

of Xwe173 was increased from 55∘C to 60∘C to increasethe specificity At the increased annealing temperatures sixpositive BAC clones were identified for the tested markerproving the suitability of the BAC library for cloningYr26 andother genes

Although BAC libraries representing direct diploidsources of wheat A genome (T monococcum [49] T urartu[50] andD genome (T tauschii) [50]) have been constructedthey cannot be used to clone the stripe rust resistance geneYr26 because it is located on wheat chromosome 1B [8 10ndash13] BAC libraries for tetraploid wheat and hexaploid wheat[29ndash35] and all chromosome-specific BAC libraries of thehexaploid cultivar Chinese Spring have been developed [3745 51ndash55] but these BAC libraries are not suitable for cloningYr26 because the gene is probably derived from T turgidumcvsdot12057480-1 the original wheat parent of 92R137 [11] ThereforeBAC libraries constructed with different cultivars are neededto clone important specific trait genes Currently we are usingthe BAC library constructed in the present study to cloneYr26to shield light to the molecular mechanisms of stripe rustresistance

Conflict of Interests

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

Acknowledgments

This study was sponsored by the National Basic ResearchProgram of China (no 2013CB127700) the National MajorProject of Breeding for New Transgenic Organisms in China(2011ZX08002-001) theNationalNatural Science Foundation

of China (31371924) and the 111 Project from the Ministry ofEducation of China (B07049)

References

[1] D Fu C Uauy A Distelfeld et al ldquoA kinase-START geneconfers temperature-dependent resistance to wheat stripe rustrdquoScience vol 323 no 5919 pp 1357ndash1360 2009

[2] X-Q Huang andM S Roder ldquoMolecular mapping of powderymildew resistance genes in wheat a reviewrdquo Euphytica vol 137no 2 pp 203ndash223 2004

[3] D Sharma-Poudyal X Chen A Wan et al ldquoVirulence char-acterization of international collections of the wheat stripe rustpathogen Puccinia striiformis f sp triticirdquo Plant Disease vol 97no 3 pp 379ndash386 2013

[4] X M Chen ldquoEpidemiology and control of stripe rust [Pucciniastriiformis f sp tritici] on wheatrdquo Canadian Journal of PlantPathology vol 27 no 3 pp 314ndash337 2005

[5] W Q Chen L RWu T G Liu et al ldquoRace dynamics diversityand virulence evolution in Puccinia striiformis f sp tritici thecausal agent of wheat stripe rust in china from 2003 to 2007rdquoPlant Disease vol 93 no 11 pp 1093ndash1101 2009

[6] G Robbelen and E L Sharp ldquoMode of inheritance interactionand application of genes conditioning resistance to yellow rustrdquoFortschritte der Pflanzenzuechtung vol 9 pp 1ndash88 1978

[7] R F Line andX Chen ldquoSuccesses in breeding for andmanagingdurable resistance to wheat rustsrdquo Plant Disease vol 79 no 12pp 1254ndash1255 1995

[8] C Wang Y Zhang D Han et al ldquoSSR and STS markers forwheat stripe rust resistance gene Yr26rdquo Euphytica vol 159 no3 pp 359ndash366 2008

[9] A Cao L Xing X Wang et al ldquoSerinethreonine kinase geneStpk-V a keymember of powderymildew resistance gene Pm21confers powderymildew resistance in wheatrdquo Proceedings of the


National Academy of Sciences of theUnited States of America vol108 no 19 pp 7727ndash7732 2011

[10] G Q Li Z F Li W Y Yang et al ldquoMolecular mapping ofstripe rust resistance gene YrCH42 in Chinese wheat cultivarChuanmai 42 and its allelism with Yr24 and Yr26rdquo Theoreticaland Applied Genetics vol 112 no 8 pp 1434ndash1440 2006

[11] W E Wen G Q Li Z H He W Y Yang M L Xu andX C Xia ldquoDevelopment of an STS marker tightly linked toYr26 against wheat stripe rust using the resistance gene-analogpolymorphism (RGAP) techniquerdquoMolecular Breeding vol 22no 4 pp 507ndash515 2008

[12] X Zhang D Han Q Zeng et al ldquoFine mapping of wheat striperust resistance gene Yr26 based on collinearity of wheat withBrachypodium distachyon and ricerdquo PLoS ONE vol 8 no 3Article ID e57885 2013

[13] J Ma R Zhou Y Dong L Wang X Wang and J JialdquoMolecular mapping and detection of the yellow rust resistancegene Yr26 in wheat transferred from Triticum turgidum L usingmicrosatellite markersrdquo Euphytica vol 120 no 2 pp 219ndash2262001

[14] T G Liu Y L Peng W Q Chen et al ldquoFirst detection ofvirulence in Puccinia striiformis f sp tritici in China to resis-tance genes Yr24 (=Yr26) present in wheat cultivar chuanmai42rdquo Plant Disease vol 94 no 9 pp 1163ndash1163 2010

[15] Q-D Zeng D-J Han Q-L Wang et al ldquoStripe rust resistanceand genes in Chinese wheat cultivars and breeding linesrdquoEuphytica vol 196 no 2 pp 271ndash284 2014

[16] S Cloutier B D McCallum C Loutre et al ldquoLeaf rust resis-tance gene Lr1 isolated from bread wheat (Triticum aestivumL) is amember of the large psr567 gene familyrdquo PlantMolecularBiology vol 65 no 1-2 pp 93ndash106 2007

[17] C Feuillet S Travella N Stein L Albar A Nublat and BKeller ldquoMap-based isolation of the leaf rust disease resistancegene Lr10 from the hexaploid wheat (Triticum aestivum L)genomerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 25 pp 15253ndash15258 2003

[18] L Huang S A Brooks W Li J P Fellers H N Trick and B SGill ldquoMap-based cloning of leaf rust resistance gene Lr21 fromthe large and polyploid genome of bread wheatrdquo Genetics vol164 no 2 pp 655ndash664 2003

[19] S Periyannan J Moore M Ayliffe et al ldquoThe Gene Sr33 anortholog of barley mla genes encodes resistance to wheat stemrust race Ug99rdquo Science vol 341 no 6147 pp 786ndash788 2013

[20] C Saintenac W Zhang A Salcedo et al ldquoIdentification ofwheat gene Sr35 that confers resistance to Ug99 stem rust racegrouprdquo Science vol 341 no 6147 pp 783ndash786 2013

[21] S G Krattinger E S Lagudah W Spielmeyer et al ldquoA putativeABC transporter confers durable resistance to multiple fungalpathogens in wheatrdquo Science vol 323 no 5919 pp 1360ndash13632009

[22] K Farrar and I S Donnison ldquoConstruction and screening ofBAC librariesmade fromBrachypodium genomicDNArdquoNatureProtocols vol 2 no 7 pp 1661ndash1674 2007

[23] H B Zhang X Zhao X Ding A H Paterson and R A WingldquoPreparation of megabase-size DNA from plant nucleirdquo PlantJournal vol 7 no 1 pp 175ndash184 1995

[24] S J Strong Y Ohta G W Litman and C T AmemiyaldquoMarked improvement of PAC and BAC cloning is achievedusing electroelution of pulsed-field gel-separated partial digestsof genomic DNArdquo Nucleic Acids Research vol 25 no 19 pp3959ndash3961 1997

[25] R R Klein D T Morishige P E Klein J Dong and J EMullet ldquoHigh throughput BACDNA isolation for physical mapconstruction of sorghum (Sorghum bicolor)rdquo Plant MolecularBiology Reporter vol 16 no 4 pp 351ndash364 1998

[26] K Osoegawa P YWoon B Zhao et al ldquoAn improved approachfor construction of bacterial artificial chromosome librariesrdquoGenomics vol 52 no 1 pp 1ndash8 1998

[27] D G Peterson J P Tomkins D A Frisch et al ldquoConstructionof plant bacterial artificial chromosome (BAC) libraries anillustrated guiderdquo Journal of Agricultural Genomics vol 5 pp1ndash100 2000

[28] M Luo and R A Wing ldquoAn improved method for plant BAClibrary constructionrdquo Methods in Molecular Biology vol 236pp 3ndash20 2003

[29] ZMa SWeining P J Sharp and C Liu ldquoNon-gridded librarya new approach for BAC (bacterial artificial chromosome)exploitation in hexaploid wheat (Triticum aestivum)rdquo NucleicAcids Research vol 28 no 24 article E106 2000

[30] S Allouis G Moore A Bellec et al ldquoConstruction andcharacterisation of a hexaploid wheat (Triticum aestivum L)BAC library from the reference germplasm lsquoChinese SpringrsquordquoCereal Research Communications vol 31 no 3-4 pp 331ndash3382003

[31] A Cenci N Chantret X Kong et al ldquoConstruction andcharacterization of a half million clone BAC library of durumwheat (Triticum turgidum ssp durum)rdquoTheoretical and AppliedGenetics vol 107 no 5 pp 931ndash939 2003

[32] S D Nilmalgoda S Cloutier and A Z Walichnowski ldquoCon-struction and characterization of a bacterial artificial chro-mosome (BAC) library of hexaploid wheat (Triticum aestivumL) and validation of genome coverage using locus-specificprimersrdquo Genome vol 46 no 5 pp 870ndash878 2003

[33] P Ling and X M Chen ldquoConstruction of a hexaploid wheat(Triticum aestivum L) bacterial artificial chromosome libraryfor cloning genes for stripe rust resistancerdquoGenome vol 48 no6 pp 1028ndash1036 2005

[34] I Ratnayaka M Baga D B Fowler and R N ChibbarldquoConstruction and characterization of a BAC library of a cold-tolerant hexaploid wheat cultivarrdquo Crop Science vol 45 no 4pp 1571ndash1577 2005

[35] B Shen D M Wang C L McIntyre and C J Liu ldquoA ldquoChineseSpringrdquo wheat (Triticum aestivum L) bacterial artificial chro-mosome library and its use in the isolation of SSR markers fortargeted genome regionsrdquoTheoretical and Applied Genetics vol111 no 8 pp 1489ndash1494 2005

[36] M D Bennett and I J Leitch ldquoNuclear DNA amounts inangiospermsrdquo Annals of Botany vol 76 no 2 pp 113ndash176 1995

[37] H Simkova J Safar M Kubalakova et al ldquoBAC libraries fromwheat chromosome 7D efficient tool for positional cloning ofaphid resistance genesrdquo Journal of Biomedicine and Biotechnol-ogy vol 2011 Article ID 302543 11 pages 2011

[38] B J Shi T Sutton N C Collins M Pallotta and P LangridgeldquoConstruction of a barley bacterial artificial chromosomelibrary suitable for cloning genes for boron tolerance sodiumexclusion and high grain zinc contentrdquo Plant Breeding vol 129no 3 pp 291ndash296 2010

[39] D Schulte R Ariyadasa B Shi et al ldquoBAC library resourcesfor map-based cloning and physical map construction in barley(Hordeum vulgare L)rdquo BMC Genomics vol 12 article 247 2011


[40] F M You M-C Luo K Xu K R Deal O D Anderson andJ Dvorak ldquoA new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screeningrdquoBMC Genomics vol 11 no 1 article 692 2010

[41] A M Atrazhev and J F Elliott ldquoSimplified desalting of ligationreactions immediately prior to electroporation into E colirdquoBioTechniques vol 21 no 6 article 1024 1996

[42] Q Tao Y-L Chang J Wang et al ldquoBacterial artificialchromosome-based physical map of the rice genome con-structed by restriction fingerprint analysisrdquo Genetics vol 158no 4 pp 1711ndash1724 2001

[43] N Huo Y Q Gu G R Lazo et al ldquoConstruction and charac-terization of two BAC libraries from Brachypodium distachyona new model for grass genomicsrdquo Genome vol 49 no 9 pp1099ndash1108 2006

[44] T N Foote S Griffiths S Allouis andGMoore ldquoConstructionand analysis of a BAC library in the grass Brachypodiumsylvaticum its use as a tool to bridge the gap between rice andwheat in elucidating gene contentrdquo Functional and IntegrativeGenomics vol 4 no 1 pp 26ndash33 2004

[45] J Safar H Simkova M Kubalakova et al ldquoGeneratingresources for genomics of wheat homoeologous chromosomegroup 3 3AS- and 3DS-specific BAC librariesrdquo Journal ofGenetics and Breeding vol 61 no 1-2 pp 151ndash160 2007

[46] B Chalhoub H Belcram and M Caboche ldquoEfficient cloningof plant genomes into bacterial artificial chromosome (BAC)libraries with larger and more uniform insert sizerdquo PlantBiotechnology Journal vol 2 no 3 pp 181ndash188 2004

[47] E Barillot B Lacroix and D Cohen ldquoTheoretical analysisof library screening using a N-dimensional pooling strategyrdquoNucleic Acids Research vol 19 no 22 pp 6241ndash6247 1991

[48] W J Bruno E Knill D J Balding et al ldquoEfficient poolingdesigns for library screeningrdquo Genomics vol 26 no 1 pp 21ndash30 1995

[49] D Lijavetzky G Muzzi T Wicker B Keller R Wing and JDubcovsky ldquoConstruction and characterization of a bacterialartificial chromosome (BAC) library for the A genome ofwheatrdquo Genome vol 42 no 6 pp 1176ndash1182 1999

[50] E D Akhunov A R Akhunova and J Dvorak ldquoBAC librariesof Triticum urartu Aegilops speltoides and Ae tauschii thediploid ancestors of polyploid wheatrdquo Theoretical and AppliedGenetics vol 111 no 8 pp 1617ndash1622 2005

[51] J Janda J Bartos J Safar et al ldquoConstruction of a subgenomicBAC library specific for chromosomes 1D 4D and 6D ofhexaploid wheatrdquoTheoretical and Applied Genetics vol 109 no7 pp 1337ndash1345 2004

[52] J Safar J Bartos J Janda et al ldquoDissecting large and complexgenomes flow sorting and BAC cloning of individual chromo-somes from bread wheatrdquo Plant Journal vol 39 no 6 pp 960ndash968 2004

[53] J Janda J Safar M Kubalakova et al ldquoAdvanced resourcesfor plant genomics a BAC library specific for the short arm ofwheat chromosome 1Brdquo Plant Journal vol 47 no 6 pp 977ndash986 2006

[54] E Paux D Roger E Badaeva et al ldquoCharacterizing the com-position and evolution of homoeologous genomes in hexaploidwheat through BAC-end sequencing on chromosome 3Brdquo PlantJournal vol 48 no 3 pp 463ndash474 2006

[55] J Safar H Simkova M Kubalakova et al ldquoDevelopment ofchromosome-specific BAC resources for genomics of breadwheatrdquo Cytogenetic and Genome Research vol 129 no 1-3 pp211ndash223 2010

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


caused by Blumeria graminis f sp tritici (Bgt) [9] It has astripe rust resistance gene Yr26 which is effective againstpredominant races of Pst in China [8 10ndash13] Although Yr26is identified as susceptible to a new virulent pathotype it canprovide good protection of crops from the Pst populationwhen combined with other resistance genes because thevirulent pathotype has a narrow spectrum of virulence[14] Because 92R137 has resistance to both stripe rust andpowdery mildew the two most important wheat diseasesin China the line has been widely used in wheat breedingprograms in China [8] Cloning Yr26 can contribute tothe understanding of molecular mechanisms of stripe rustresistance and provide basis for wisely using the gene andother genes to achieve long lasting and high level resistancesto stripe rust

A high-resolution map has been developed for Yr26 ina population developed with 92R137 [14] Among a largenumber of markers linked with Yr26 codominant markerXwe173 which was developed from wheat EST BF474347 [8]was found to be only 14 cM away and appeared to be reliablefor marker-assisted detection of the gene [15] Because Yr26is mapped close to the centromere of chromosome 1B [8] it isdifficult to identify closer markers Although we are currentlyusing a much larger population (composed of nearly 10000F2individuals) for identifying closer markers a bacterial

artificial chromosome library (BAC) developed from theresistant wheat line should speed up the process of cloningYr26

In Triticeae seven genes for resistance to rusts are clonedincluding Lr1 [16] Lr10 [17] and Lr21 [18] for resistance toleaf rust Sr33 [19] and Sr35 [20] for resistance to stem rustYr36 [1] for resistance to stripe rust and Lr34Yr18 conferringresistance to both leaf rust and stripe rust [21] These molec-ularly very diverse genes were cloned all using themap-basedcloning approach Therefore we have taken the approach forcloning Yr26 in 92R137 This approach requires establishingphysicalmaps using a large insert library Among the differentkinds of libraries a BAC library is a preferred system overother large insert libraries for its advantage of easy operationhigh stability and low chimerism [22]

Although several protocols of BAC library construction[22ndash28] and BAC libraries for tetraploid wheat and hexaploidwheat [29ndash35] have been published research progress fordevelopment of large insertion libraries for a large genomespecies is still limited Particularly hexaploidwheat has a verylarge genome of 167 Gb [36] In various reports differentmethods were adopted to improve transformation efficiencyand quality such as multiple selection to remove small DNAmolecules [37] electroelution means to recover size-selectedDNA from agarose gel [24] and using TAE instead of TBEbuffer in electroelution [26 38] In the present study we triedto integrate almost all these modified methods to construct ahigh quality BAC library for 92R137 aimed at cloningYr26 Toimprove genome representation and reduce the gaps betweenBAC contigs resulting from uneven distribution of restrictionsites [39]HindIII and BamHI were used separately to obtainBAC clones To speed up screening the BAC for 92R137 a new5D clone pooling scheme [40] was adopted and proven to bemore efficient

2 Materials and Methods

21 Plant Material and HMW DNA Preparation Springwheat 92R137 kindly provided by Professor Peidu Chen attheNanjingAgricultural University was used for BAC libraryconstruction Seeds were sown in pots filled with soil mixtureand plants were grown in dark at room temperature Whenthey grewup to 10 cm long seedlingswere harvested forDNAextraction

Twenty-five gram leaf tissue was ground into fine powderin liquid nitrogen following the slightly modified proceduredescribed by Luo and Wing [28] to release their intactnuclei The nuclei were isolated and purified four times withice-cold NIBM (10mM Tris-HCl with a pH of 80 10mMEDTA with a pH of 80 100mM KCl 05M sucrose 4mMspermidine 1mM spermine with 10 Triton X-100 and01 120573-mercaptoethanol) at 10mL1 g tissue and centrifugedat a force set to 1800 g The obtained pellet was gentlyresuspended in approx 1mL NIB (10mM Tris-HCl pH 8010mM EDTA pH of 80 100mM KCl 05M sucrose 4mMspermidine and 1mM spermine with 10 Triton X-100)At the equal volume 1 (wv) low melting temperature(LMP) agarose (Amresco Solon OH USA) dissolved in NIBwas added into the suspension and the mixture was pouredinto a 100120583L Plug Mold (Bio-Rad Hercules CA USA)After adding lysis buffer 05molL EDTA with a pH of 901 (wv) N-lauroylsarcosine and proteinase K at 1mgmL(Amresco) the plugs were rotated in an incubator at 50∘Cfor about 48 h and at 24 h the buffer was changed with freshbuffer Then the agarose plugs were rinsed twice with ice-cold T

10E10

buffer (10mmolL Tris-HCl with a pH of 8010mmolL EDTA with a pH of 80) containing 1mmolLphenylmethylsulphonyl fluoride (Sigma St Louis MO USA)and twice with ice-cold TE buffer 1 hour each rinse Thenthe plugs were stored in TE buffer (pH 80) at 4∘C for futureuse

22 Restriction Digestion and DNA Fragment Size SelectionTo determine the optimum conditions for the highest per-centage of fragments between 120 and 2425 kb a series ofeight partial digests were performed One half of the DNAplug was chopped into fine pieces as one treatment andequilibrated with 45120583L of restriction enzyme buffer for 30minutes 5 120583L enzyme diluents with 0 04 08 12 16 20 24and 10U BamHI or HindIII (New England Biolabs IpswichMA USA) were added into the treatment contained inmicrocentrifuge tubes and placed on ice for 30minutesThenthe microcentrifuge tubes were incubated in 37∘C water bathfor 30 minutes for digestion After adding 10 120583L 05molLEDTA (pH 80) the tubes were incubated on ice for at least10min to stop digestion

The plug pieces were separated in a 1 agarose gel in 05xTBE using aCHEFDR-III System (Bio-Rad) at 6Vcm 1ndash50 sswitch times linear ramp 120∘ angle at 14∘C for 18 h Once theoptimal digestion condition for one batch of plugs to producefragments between 120 and 2425 kbwas identified the partialdigestions of 6 plugs were performed under the optimaldigestion condition DNA fragments with sizes ranging from120 to 2425 kb were excised (corresponding to 120ndash180 and














Lane

485

97

1455

1942425

2913395

388

(kb)


(a)

485

97

1455

19424252913395388

(kb)


(b)










3 Results



(kb)

14551942425291

97485

(a)

485

97

1455194

(kb)

(b)





48597

1455194

2425

(kb)

M

Vector





02468

10121416

Prop

ortio

n of

diff

eren

t ins

ert

Insert size (kb)


size c

lone

s (

)

0

0ndash10

10

ndash20

20

ndash30

30

ndash40

40

ndash50

50

ndash60

60

ndash70

70

ndash80

80

ndash90

90

ndash100

100

ndash110

110

ndash120

120

ndash130

130

ndash140

140

ndash150

150

ndash160

160

ndash170

170

ndash180

180

ndash190

190

ndash200

gt200




4 Discussion








92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)






Acknowledgments



References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Lane

485

97

1455

1942425

2913395

388

(kb)


(a)

485

97

1455

19424252913395388

(kb)


(b)










3 Results



(kb)

14551942425291

97485

(a)

485

97

1455194

(kb)

(b)





48597

1455194

2425

(kb)

M

Vector





02468

10121416

Prop

ortio

n of

diff

eren

t ins

ert

Insert size (kb)


size c

lone

s (

)

0

0ndash10

10

ndash20

20

ndash30

30

ndash40

40

ndash50

50

ndash60

60

ndash70

70

ndash80

80

ndash90

90

ndash100

100

ndash110

110

ndash120

120

ndash130

130

ndash140

140

ndash150

150

ndash160

160

ndash170

170

ndash180

180

ndash190

190

ndash200

gt200




4 Discussion








92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)






Acknowledgments



References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(kb)

14551942425291

97485

(a)

485

97

1455194

(kb)

(b)





48597

1455194

2425

(kb)

M

Vector





02468

10121416

Prop

ortio

n of

diff

eren

t ins

ert

Insert size (kb)


size c

lone

s (

)

0

0ndash10

10

ndash20

20

ndash30

30

ndash40

40

ndash50

50

ndash60

60

ndash70

70

ndash80

80

ndash90

90

ndash100

100

ndash110

110

ndash120

120

ndash130

130

ndash140

140

ndash150

150

ndash160

160

ndash170

170

ndash180

180

ndash190

190

ndash200

gt200




4 Discussion








92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)






Acknowledgments



References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


02468

10121416

Prop

ortio

n of

diff

eren

t ins

ert

Insert size (kb)


size c

lone

s (

)

0

0ndash10

10

ndash20

20

ndash30

30

ndash40

40

ndash50

50

ndash60

60

ndash70

70

ndash80

80

ndash90

90

ndash100

100

ndash110

110

ndash120

120

ndash130

130

ndash140

140

ndash150

150

ndash160

160

ndash170

170

ndash180

180

ndash190

190

ndash200

gt200




4 Discussion








92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)






Acknowledgments



References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


92R1

37

B634

92R1

37

H18

6

451bp

451bp

(a)

92R1

37

H18

6H13

H18

6E15

B634

C11

B634

C10

B634

D10

B6

34E1

0M M

(b)






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

research article construction and characterization of a...

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